Ligand for G-protein coupled receptor FPRL2

ABSTRACT

The present invention relates to methods, reagents and kits for detecting of formyl peptide receptor like-2 (FPRL2) polypeptide activity in a sample and identifying agents which modulate polypeptide activity. It further relates to antibodies raised against FPRL2. It further relates to substances for preventing, treating and/or alleviating diseases or disorders characterized by dysregulation of FPRL2 polypeptide signalling.

RELATED APPLICATION(S)

This is a divisional patent application of U.S. patent application Ser.No. 11/129,107, filed May 13, 2005, now U.S. Pat. No. 7,582,416 whichclaims the benefit of European Patent Application 04447122.5, filed May14, 2004 and European Patent Application 04447231.4, filed Oct. 18,2004. The entire teachings of the above application(s) are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention is related to the natural ligand for an orphan Gprotein coupled receptor FPRL2 and methods of use. It further relates toantibodies raised against FPRL2.

BACKGROUND

G-protein coupled receptors (GPCRs) are proteins responsible fortransducing a signal within a cell. GPCRs have usually seventransmembrane domains. Upon binding of a ligand to an extra-cellularportion or fragment of a GPCR, a signal is transduced within the cellthat results in a change in a biological or physiological property orbehaviour of the cell. GPCRs, along with G-proteins and effectors(intracellular enzymes and channels modulated by G-proteins), are thecomponents of a modular signalling system that connects the state ofintra-cellular second messengers to extra-cellular inputs.

GPCR genes and gene products can modulate various physiologicalprocesses and are potential causative agents of disease. The GPCRs seemto be of critical importance to both the central nervous system andperipheral physiological processes.

The GPCR protein superfamily is represented by five families: Family I,receptors typified by rhodopsin and the beta2-adrenergic receptor andcurrently represented by over 200 unique members; Family II, theparathyroid hormone/calcitonin/secretin receptor family; Family III, themetabotropic glutamate receptor family, Family IV, the CAMP receptorfamily, important in the chemotaxis and development of D. discoideum;and Family V, the fungal mating pheromone receptor such as STE2.

G proteins represent a family of heterotrimeric proteins composed of α,β and γ subunits, that bind guanine nucleotides. These proteins areusually linked to cell surface receptors (receptors containing seventransmembrane domains) for signal transduction. Indeed, following ligandbinding to the GPCR, a conformational change is transmitted to the Gprotein, which causes the α-subunit to exchange a bound GDP molecule fora GTP molecule and to dissociate from the βγ-subunits.

The GTP-bound form of the α, β and γ-subunits typically functions as aneffector-modulating moiety, leading to the production of secondmessengers, such as cAMP (e.g. by activation of adenyl cyclase),diacylglycerol or inositol phosphates.

More than 20 different types of α-subunits are known in humans. Thesesubunits associate with a small pool of β and γ subunits. Examples ofmammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins aredescribed extensively in Lodish et al., Molecular Cell Biology(Scientific American Books Inc., New York, N.Y., 1995; and also byDownes and Gautam, 1999, The G-Protein Subunit Gene Families. Genomics62:544-552), the contents of both of which are incorporated herein byreference.

Known and uncharacterized GPCRs currently constitute major targets fordrug action and development. There are ongoing efforts to identify new Gprotein coupled receptors which can be used to screen for new agonistsand antagonists having potential prophylactic and therapeuticproperties.

More than 300 GPCRs have been cloned to date, excluding the family ofolfactory receptors. Mechanistically, approximately 50-60% of allclinically relevant drugs act by modulating the functions of variousGPCRs (Cudermann et al., J. Mol. Med., 73:51-63, 1995).

Formyl peptide receptor-like 2 (FPRL2) (SEQ ID NO: 1, humanpolynucleotide sequence, SEQ ID NO: 2, human amino acid sequence) is amember of FPR Family. The members of this family belong to the GPCRfamily. Human FPR (SEQ ID NO: 3, human polynucleotide sequence, SEQ IDNO: 4, human amino acid sequence) was first member of the FRP familydefined biochemically, in 1976, as a high affinity binding site on thesurface of neutrophils for the prototypic N-formyl peptideformyl-methionine-leucyl-phenylalanine (fMLF). It was then cloned in1990, by Boulay et al. from a differentiated HL-60 myeloid leukemia-cellcDNA library [Boulay, F. et al. (1990) Biochem. Biophys. Res. Commun.168, 1103-1109; Boulay, F. et al. (1990) Biochemistry 29, 11123-11133].In transfected cell lines, FPR binds fMLF with high affinity (K_(d)<1nM) and is activated by picomolar to low nanomolar concentrations offMLF in chemotaxis and calcium ion (Ca²⁺) mobilization assays.

Two additional human genes, designated FPRL1 (FPR-like 1) (SEQ ID NO: 5,human polynucleotide sequence; SEQ ID NO: 6, human amino acid sequence)and FPRL2 (FPR-like 2), were subsequently isolated by low-stringencyhybridization using FPR cDNA as a probe [Ye, R. D. et al. (1992)Biochem. Biophys. Res. Commun. 184, 582-589; Bao, L. et al. (1992)Genomics. 13, 437-440] and shown to cluster with FPR on human chromosome19q13.3 [Murphy, P. M. et al. (1992) J. Biol. Chem. 267, 7637-7643; Bao,L. et al. (1992) Genomics 13, 437-440]. FPRL1 is defined as alow-affinity fMLF receptor, based on its activation only by highconcentrations of Fmlf (μM range) in vitro [Murphy, P. M. (1996)Chemoattractant Ligands and their Receptors (Horuk R, ed.), pp. 269-299,CRC Press, Inc., Boca Raton; Prossnitz, E. R. and Ye, R. D. (1997)Pharmacol. Ther. 74, 73-102]. However, it is unclear whether suchconcentrations of fMLF could be generated at sites of bacterialinfection or tissue injury. Therefore, the role of FPRL1 as another bonafide functional fMLF receptor in vivo remains to be determined. FPRL2does not bind or respond to N-formyl peptides [Durstin, M. et al. (1994)Biochem. Biophys. Res. Commun. 201, 174-179] but instead shares somenon-formylated chemotactic peptides identified for FPRL1 [Christophe, T.et al. (2001) J. Biol. Chem. 276, 21585-21593; Betten, A. et al. (2001)J. Clin. Invest. 108, 1221-1228].

Although FPR and FPRL1 were initially detected in phagocytic leukocytes,other cell types also express these receptors but with undefinedbiological significance. Little information is available about theexpression pattern of FPRL2, except that mRNA for this receptor ispresent in monocytes but not neutrophils [Durstin, M. et al. (1994)Biochem. Biophys. Res. Commun. 201, 174-179]. Functional FPRL2 is alsoexpressed in mature dentritic cells (DCs) [Yang, D. et al. J. Leukoc.Biol. Vol. 72: 598-607 (2002)], which express reduced levels of FPR butdo not appear to express FPRL1 [Yang, D. et al. (2001) J. Immunol. 166,4092-4098; Braun, M. C. et al. (2001) Blood 97, 3531-3536].

The Heme Binding Protein (HBP) (Sequence ID No7: human polynucleotidesequence, Sequence ID No8: human amino acid sequence; Sequence ID No9:mouse polynucleotide sequence, Sequence ID No10: mouse amino acidsequence). The human and mouse HBP cDNAs are 567 and 570 bp longrespectively and encode a protein product of 189 and 190 amino acidsrespectively. This protein is located into the cytoplasm of the cell.HPB binds heme and porphyrins with micromolar Kd. HBP may function as abuffer for overproduced porphyrin as well as heme. Expression studiesindicated that the mouse mRNA encoding HBP is expressed in liver, spleenand kidney cells (Blackmon et al; 2002 Arch. of Biochem. and Biophysics407, p 196-201).

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for detecting FPRL2polypeptide activity in a sample comprising the steps of:

-   -   a) incubating a sample comprising FPRL2 polypeptide with HBP        polypeptide under conditions which permit binding of FPRL2        polypeptide and HBP polypeptide, and    -   b) detecting a second messenger.

Another embodiment of the present invention is a method as describedabove further comprising the steps of:

-   -   a) incubating a second sample comprising FPRL2 polypeptide in        the absence of HBP polypeptide under conditions which permit        binding of FPRL2 polypeptide and HBP polypeptide, and    -   b) detecting a second messenger.

Another embodiment of the present invention is a method as describedabove wherein said sample comprises cells expressing FPRL2 polypeptide.

Another embodiment of the present invention is a method as describedabove wherein said sample comprises cell membranes bearing FPRL2polypeptide.

Another embodiment of the present invention is a method as describedabove wherein said incubating is performed in or on virus-inducedbudding membranes containing a FPRL2 polypeptide polypeptide.

Another embodiment of the present invention is a method as describedabove, wherein step a) is further performed in the presence of Gα16polypeptide.

Another embodiment of the present invention is a method of identifyingan agent that binds to FPRL2 polypeptide, said method comprising:

-   -   (a) contacting a FPRL2 polypeptide with HBP polypeptide in the        presence or absence of a candidate binding agent under        conditions permitting binding of said HBP polypeptide to said        FPRL2 polypeptide; and    -   (b) measuring binding of said FPRL2 polypeptide to said HBP        polypeptide, wherein a decrease in binding in the presence of        said candidate binding agent, relative to binding in the absence        of said candidate binding agent, identifies said candidate        binding agent as an agent that binds to FPRL2 polypeptide.

Another embodiment of the present invention is a method as describedabove, wherein said agent is present in a sample.

Another embodiment of the present invention is a method of identifyingan agent that increases the signaling activity of FPRL2 polypeptide,said method comprising:

-   -   (a) contacting a FPRL2 polypeptide with an agent;    -   (b) measuring a signaling activity of said FPRL2 polypeptide in        the presence of said agent; and    -   (c) comparing said activity measured in the presence of said        agent to said activity measured in a reaction in which said        FPRL2 polypeptide is contacted with HBP polypeptide, wherein        said agent is identified as an agonist that increases the        signaling of said FPRL2 polypeptide when the amount of said        activity measured in the presence of said agent is at least 10%        of the amount induced by said HBP polypeptide.

Another embodiment of the present invention is a method as describedabove, wherein said agent is present in a sample.

Another embodiment of the present invention is a method of identifyingan agent that decreases the signaling activity of FPRL2 polypeptide,said method comprising:

-   -   (a) contacting a FPRL2 polypeptide with HBP polypeptide in the        presence or absence of said agent;    -   (b) measuring a signaling activity of said FPRL2 polypeptide;    -   (c) comparing the amount of said activity measured in a reaction        containing FPRL2 polypeptide and said HBP polypeptide without        said agent to the amount of said activity measured in a reaction        containing said FPRL2 polypeptide, said HBP polypeptide and said        agent, wherein a decrease in said activity in the presence of        said agent relative to the activity in the absence of said agent        identifies said agent as an antagonist or inverse agonist for        said FPRL2 polypeptide.

Another embodiment of the present invention is a method as describedabove, wherein said agent is present in a sample.

Another embodiment of the present invention is a method as describedabove wherein said FPRL2 polypeptide is expressed by cells on theirsurface.

Another embodiment of the present invention is a method as describedabove wherein said FPRL2 polypeptide is present in cell membranes.

Another embodiment of the present invention is a method as describedabove, wherein said FPRL2 polypeptide is present in or on virus-inducedbudding membranes.

Another embodiment of the present invention is a method as describedabove wherein said cells are selected from the group consisting of:COS7-cells, a CHO cell, a LM (TK-) cell, a NIH-3T3 cell, HEK-293 cell,K-562 cell and a 1321N1 astrocytoma cell and other cell lines.

Another embodiment of the present invention is a method as describedabove, further performed in the presence of Gα16 polypeptide.

Another embodiment of the present invention is a method as describedabove wherein said measuring or said detecting is performed using amethod selected from label displacement, surface plasmon resonance,fluorescence resonance energy transfer, fluorescence quenching, andfluorescence polarization.

Another embodiment of the present invention is a method as describedabove wherein said agent is selected from the group consisting of anatural or synthetic peptide, a polypeptide, an antibody orantigen-binding fragment thereof, a lipid, a carbohydrate, a nucleicacid, and a small organic molecule.

Another embodiment of the present invention is a method as describedabove wherein said detecting or measuring a signalling activity ormeasuring the binding of said FPRL2 polypeptide comprises detecting achange in the level of a second messenger.

Another embodiment of the present invention is a method as describedabove wherein the step of detecting a signalling activity or saidmeasuring a signalling activity or measuring the binding comprisesmeasurement of guanine nucleotide binding or exchange, adenylate cyclaseactivity, cAMP, protein kinase C activity, phosphatidylinositolbreakdown, diacylglycerol, inositol trisphosphate, intracellularcalcium, arachinoid acid concentration, MAP kinase activity, tyrosinekinase activity, reporter gene expression.

Another embodiment of the present invention is a method as describedabove wherein said measuring a signalling activity comprises using anaequorin-based assay.

Another embodiment of the present invention is an agent obtainable usinga screening method disclosed herein.

Another embodiment of the present invention is an antibody whichspecifically reacts with FPRL2 polypeptide and which increases ordecreases:

-   -   (a) the binding of HBP polypeptide to the FPRL2 polypeptide, or    -   (b) the signalling activity of HBP polypeptide bound to the        FPLRL2 polypeptide

Another embodiment of the present invention is a method of in vitrodiagnosing a disease or disorder characterized by dysregulation of FPRL2polypeptide signalling, said method comprising:

-   -   a) contacting a tissue sample comprising a FPRL2 polypeptide        with HBP polypeptide;    -   b) detecting binding of said HBP polypeptide to said tissue        sample; and    -   c) comparing the binding detected in step (b) with a standard,        wherein a difference in binding relative to said standard is        diagnostic of a disease or disorder characterized by        dysregulation of FPRL2 polypeptide signalling.

Another embodiment of the present invention is a method of in vitrodiagnosing a disease or disorder characterized by dysregulation of FPRL2polypeptide signalling, said method comprising:

-   -   a) contacting a tissue sample comprising a FPRL2 polypeptide        with HBP polypeptide;    -   b) detecting a signalling activity of FPRL2 polypeptide in said        tissue sample; and    -   c) comparing the signalling activity detected in step (b) with a        standard, wherein a difference in signalling activity relative        to said standard is diagnostic of a disease or disorder        characterized by dysregulation of FPRL2 polypeptide signalling.

Another embodiment of the present invention is a method as describedabove wherein said comparing is performed on a microarray.

Another embodiment of the present invention is a kit for detectingbinding to FPRL2 polypeptide, an agent binding to FPRL2 polypeptide oran agent decreasing or increasing the signalling activity of FPRL2polypeptide, said kit comprising a FPRL2 polypeptide and HBPpolypeptide, and packaging materials therefore, wherein said FPRL2polypeptide and HBP polypeptide are packaged separately.

Another embodiment of the present invention is a kit as described above,wherein said FPRL2 polypeptide is present in a cell expressing FPRL2polypeptide and wherein said kit further comprises an antibody specificfor FPRL2 polypeptide or a FPRL2 polypeptide-specific nucleic probepackaged separately.

Another embodiment of the present invention is a kit as described above,wherein said cell is selected from the group consisting of: COS7-cells,a CHO cell, a LM (TK-) cell, a NIH-3T3 cell, HEK-293 cell, K-562 celland a 1321N1 astrocytoma cell and other cell lines.

Another embodiment of the present invention is a kit as described above,wherein said FPRL2 polypeptide is present in an isolated cell membranebearing FPRL2 polypeptide.

Another embodiment of the present invention is a kit as described above,said kit further comprising one or more components of a second messengerassay.

Another embodiment of the present invention is a kit as described above,said kit further comprising Gα16 polypeptide.

Another embodiment of the present invention is a kit for screening foragents that increase or decrease the signalling activity of FPRL2polypeptide, said kit comprising

-   -   (a) an isolated polynucleotide encoding a FPRL2 polypeptide, HBP        polypeptide and means for detecting FPRL2 polypeptide        signalling, and packaging materials therefore, or    -   (b) a cell transformed with a polynucleotide encoding a FPRL2        polypeptide, HBP polypeptide and means for detecting FPRL2        polypeptide signalling, and packaging materials therefore.

Another embodiment of the present invention is a kit as described above,wherein the said agents are detected using an antibody specific forFPRL2 polypeptide or a FPRL2 polypeptide-specific nucleic acid probe.

Another embodiment of the present invention is a kit as described abovefor the diagnosis of a disease or disorder characterized bydysregulation of FPRL2 polypeptide signalling.

Another embodiment of the present invention is a kit as described above,wherein the said disease or disorder is detected using an antibodyspecific for FPRL2 polypeptide or a FPRL2 polypeptide-specific nucleicacid probe.

Another embodiment of the present invention is a kit as described abovefurther comprising a standard of FPRL2 polypeptide activity as measuredin a cell line expressing FPRL2 polypeptide in the presence of HBPpolypeptide.

Another embodiment of the present invention is a use of HBP polypeptide,or an antibody as described above for the manufacture of apharmaceutical composition for preventing, treating and/or alleviatingdiseases or disorders characterized by dysregulation of FPRL2polypeptide signalling.

Another embodiment of the present invention is a use as described above,wherein said diseases or disorders characterized by dysregulation ofFPRL2 polypeptide signalling are selected from the group consisting ofcell migration, cancer, development of tumours and tumour metastasis,inflammatory and neoplastic processes, wound and bone healing anddysfunction of regulatory growth functions, obesity, anorexia, bulimia,acute heart failure, hypotension, hypertension, urinary retention,osteoporosis, angina pectoris, restenosis, atherosclerosis, thrombosisand other cardiovascular diseases, autoimmune and, diseasescharacterized by excessive smooth muscle cell proliferation, aneurysms,diseases characterized by loss of smooth muscle cells or reduced smoothmuscle cell proliferation, stroke, ischemia, ulcers, allergies,prostatic hypertrophy, migraine, vomiting, psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression,depression, delirium, dementia and severe mental retardation,degenerative diseases, neurodegenerative diseases such as Alzheimer'sdisease or Parkinson's disease, and dyskinasias, such as Huntington'sdisease or Gilles de la Tourett's syndrome and other related diseasesincluding thrombosis and other cardiovascular diseases, autoimmune andinflammatory diseases such as psoriasis, Eczeme, inflammatory andtrophic diseases of skin, rheumatoid arthritis, scleroderma, lupus,polymyositis, dermatomysitis, Crohn's disease, inflammatory boweldisease (IBD), Irritable Bowel Syndrome, Ulcerative Colitis, Asthma,Chronic Obstructive Pulmonary Disease, Allergic Rhinitis, Fibromyalgia,Organ Transplant Rejection, Graft versus host disease, MultipleSclerosis, Acute, Ischemic Stroke, Infectious diseases, Hepatitis A,Hepatitis B, Hepatitis C, Sepsis, Septic shock, Chronic bronchitis,infections such as bacterial, fungal, protozoan and viral infections,such as infections caused by HIV1 and HIV2, and pain, cancer, anorexia,bulimia, asthma, acute heart failure, hypertension, urinary retention,osteoporosis, angina pectoris, myocardial infarction, ulcers, allergies,benign prostatic hypertrophy, and Type 1 Diabetes, Type 2 Diabetes,Osteoarthritis, Diabetic Retinopathy, Diabetic Nephropathy and fertilitydysfunctions, foetal developmental disorders

Another embodiment of the present invention is a method for theproduction of a pharmaceutical composition comprising the steps ofadmixing an antibody as described above, with a pharmaceutical carrier.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising an antibody as described above.

Another embodiment of the present invention is a method, kit, use orantibody as described above wherein an IBP polypeptide corresponds to asequence represented by SEQ ID NO: 18.

Another embodiment of the present invention is a method, kit, use orantibody as described above wherein an FPRL2 polypeptide corresponds toa sequence represented by SEQ ID NO: 2.

Another embodiment of the present invention is a HBP polypeptidecorresponding to the sequence represented by SEQ ID NO: 18.

Another embodiment of the present invention is a polypeptide which hasleast 50% identity to the sequence represented by SEQ ID NO: 18.

Another embodiment of the present invention is a polypeptide which is afunctional fragment of a HBP polypeptide corresponding to the sequencerepresented by SEQ ID NO: 18.

The present invention also relates to nucleic acids encoding said HBPpolypeptides as listed above.

Another embodiment of the present invention is a functional antibody orantigen-binding fragment thereof which specifically reacts with formylpeptide receptor like-2 (FPRL2) polypeptide and which increases ordecreases the signalling activity of the formyl peptide receptor like-2(FPRL2) polypeptide.

Another embodiment of the present invention is an antibody whichspecifically reacts with formyl peptide receptor like-2 (FPRL2)polypeptide, and which increases or decreases the signalling activity ofthe formyl peptide receptor like-2 (FTRL2) polypeptide.

Another embodiment of the present invention is an antibody whichspecifically reacts with formyl peptide receptor like-2 (FPRL2)polypeptide, and which increases or decreases the signalling activity ofthe formyl peptide receptor like-2 (FPRL2) polypeptide, when the amountof said activity measured in the presence of the antibody is at least10% of the amount induced by said HBP.

Another embodiment of the present invention is an antibody whichspecifically reacts with formyl peptide receptor like-2 (FPRL2)polypeptide, and is obtainable using a screening method as describedherein.

Another embodiment of the present invention is an antibody as describedherein wherein said antibody is an agonist of the formyl peptidereceptor like-2 (FPRL2) polypeptide.

Another embodiment of the present invention is an antibody as describedherein wherein said antibody is monoclonal.

Another embodiment of the present invention is an antibody as describedherein which corresponds to Mab FPRL2 422F 2B9 1C11 produced by thehybridoma cell line named FPRL2 422F 2B9 deposited under BCCM Accessionnumber: LMBP 6405CB, at BCCM/LMBP Plasmid collection, Department ofMolecular Biology, Gent University, Technologiepark 927, B-9052,Gent-Zwijnaarde, Belgium, on Apr. 28, 2005.

Another embodiment of the present invention is an antibody as describedherein which corresponds to Mab FPRL2 422F 2G3 1A10 produced by thehybridoma cell line named FPRL2 422F 2G3 deposited under BCCM Accessionnumber: LMBP 6406CB, at BCCM/LMBP Plasmid collection, Department ofMolecular Biology, Gent University, Technologiepark 927, B-9052,Gent-Zwijnaarde, Belgium, on Apr. 28, 2005.

Another embodiment of the present invention is an antibody as describedherein wherein said antibody is polyclonal.

Another embodiment of the present invention is an antibody as describedherein wherein said antibody is an antagonist of the formyl peptidereceptor like-2 (FPRL2) polypeptide.

Another embodiment of the present invention is an antibody as describedherein wherein said antibody is humanized.

Another embodiment of the present invention is a functional fragment ofan antibody as described herein.

Another embodiment of the present invention is a functional fragment asdescribed herein, which comprises the antigen binding fragment.

Another embodiment of the present invention is an homologous sequence ofthe amino acid sequence of an antibody or functional fragment asdescribed above, or of a nucleotide sequence encoding said antibody orfunctional fragment.

Another embodiment of the present invention is an antibody, functionalfragment or homologous sequence as described herein for preventing,treating and/or alleviating diseases or disorders characterized bydysregulation of formyl peptide receptor like-2 (FPRL2) polypeptidesignalling.

In another aspect of the invention, the invention provides method oftreating an individual with a diseases or disorders characterized bydysregulation of FPRL2 polypeptide signaling comprising administering atherapeutically effective dose of a modulator of a FPRL2 polypeptide.

In one embodiment, the modulator of a FPRL2 polypeptide is an antibody,functional fragment or homologous sequence as described herein.

Another embodiment of the present invention is an antibody, functionalfragment or homologous sequence as described herein, wherein saiddiseases or disorders characterized by dysregulation of formyl peptidereceptor like-2 (FPRL2) polypeptide signalling are selected from thegroup consisting of cell migration, cancer, development of tumours andtumour metastasis, inflammatory and neoplastic processes, wound and bonehealing and dysfunction of regulatory growth functions, obesity,anorexia, bulimia, acute heart failure, hypotension, hypertension,urinary retention, osteoporosis, angina pectoris, restenosis,atherosclerosis, thrombosis and other cardiovascular diseases,autoimmune and, diseases characterized by excessive smooth muscle cellproliferation, aneurysms, diseases characterized by loss of smoothmuscle cells or reduced smooth muscle cell proliferation, stroke,ischemia, ulcers, allergies, prostatic hypertrophy, migraine, vomiting,psychotic and neurological disorders, including anxiety, schizophrenia,manic depression, depression, delirium, dementia and severe mentalretardation, degenerative diseases, neurodegenerative diseases such asAlzheimer's disease or Parkinson's disease, and dyskinasias, such asHuntington's disease or Gilles de la Tourett's syndrome and otherrelated diseases including thrombosis and other cardiovascular diseases,autoimmune and inflammatory diseases such as psoriasis, Eczeme,inflammatory and trophic diseases of skin, rheumatoid arthritis,scleroderma, lupus, polymyositis, dermatomysitis, Crohn's disease,inflammatory bowel disease (IBD), Irritable Bowel Syndrome, UlcerativeColitis, Asthma, Chronic Obstructive Pulmonary Disease, AllergicRhinitis, Fibromyalgia, Organ Transplant Rejection, Graft versus hostdisease, Multiple Sclerosis, Acute, Ischemic Stroke, Infectiousdiseases, Hepatitis A, Hepatitis B, Hepatitis C, Sepsis, Septic shock,Chronic bronchitis, infections such as bacterial, fungal, protozoan andviral infections, such as infections caused by HIV1 and HIV2, and pain,cancer, anorexia, bulimia, asthma, acute heart failure, hypertension,urinary retention, osteoporosis, angina pectoris, myocardial infarction,ulcers, allergies, benign prostatic hypertrophy, and Type 1 Diabetes,Type 2 Diabetes, Osteoarthritis, Diabetic Retinopathy, DiabeticNephropathy and fertility dysfunctions, foetal developmental disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents nucleotide sequence (SEQ ID NO: 32) as cloned inpCDNA3 and deduced amino acid sequence (SEQ ID NO: 2) of the human FPRL2receptor. The start and stop codons are indicated in bold andrestriction sites for cloning (Eco RI and XbaI) are underlined.

FIG. 2 shows the tissue distribution of the human FPRL2 receptor.

FIG. 3 shows the mass spectrum of the active fraction on a Maldi Q-TOFmass spectrometer. The major monoisotopic mass is indicated by an arrow,as well as its sequence (SEQ ID NO: 18)

FIG. 4 illustrates the biological activity of the acetylated 21 aminoacid peptide corresponding to the N-terminus of porcine HBP (SEQ ID NO:18) on human FPRL2 receptor.

FIG. 5 illustrates the biological activity of the acetylated (SEQ ID NO:18) and non-acetylated (SEQ ID NO: 19) 21 amino acid peptide on humanFPRL2 receptor.

FIG. 6 shows the biological activity of the acetylated 21 amino acidpeptide (SEQ ID NO: 18) on human FPR receptor family.

FIG. 7 shows that FPRL2 receptor is coupled negatively to adenylatecyclase in presence of forskolin. Figure discloses SEQ ID NO: 18.

FIG. 8 lists amino acid and nucleic acid sequences according to theinvention (sequence listing).

FIG. 9. Purification from porcine spleen of the natural ligand of FPRL2.A porcine spleen homogenate was first fractionated by HPLC onto a Poroscolumn (Step 1). The absorbance (AU) and biological activity onFPRL2-expressing CHO-K1 cells are shown. The luminescence measured in anaequorin-based assay (black bars) was normalized to the responseobtained for 20 μM ATP. A1 (Activity 1) and A2 (Activity 2) representthe two active regions on the HPLC profile. They were processed togetheronto a C18 column (Step 2). Thereafter, A1 and A2 were purifiedseparately onto a SEC column (Step 3), a C4 column (Step 4) and for A1,a last C18 column (Step 5). The X axis is zoomed to focus on the regionof interest.

FIG. 10. Identification of F2L as a high affinity natural ligand ofFPRL2. A. Mass spectrometry analysis of the undigested fraction A2resulting from Step 4, using a Maldi Q-TOF mass spectrometer. B.Sequences corresponding to the major peaks of the mass spectra oftrypsin-digested A1 (Step 5) (SEQ ID NOS 33-34, respectively, in orderof appearance) and A2 (Step 4) (SEQ ID NOS 18 and 20, respectively, inorder of appearance) fractions, or undigested A2. All microsequencedpeptides were found to derive from the porcine heme-binding protein(HBP). The F2L peptide (A2 fraction) is amino-terminally acetylated. Ac:acetyl. C. Amino acid sequence alignment of human (SEQ ID NO: 8), mouse(SEQ ID NO: 10) and porcine (SEQ ID NO: 35) HBP. The sequencecorresponding to the F2L peptide (represented in bold) is identical inhuman and porcine HBP. The region containing tryptic peptides recoveredfrom fraction A1 is boxed (human HBP: NM 015987 ; murine HBP: NM013546). D. The A1 fraction (Step 5) was migrated onto SDS-PAGE,together with 10, 50 and 100 ng of aprotinin as standards. The gel wassilver-stained and it was estimated that the major band (6 kD) containedaround 300 ng of peptide. E. Biological activity of the A1 fraction onCHO-K1 cells expressing human FPRL2, using the aequorin-based assay. F.The A2 fraction (Step 4) was migrated onto SDS-PAGE, together with 10and 50 ng of aprotinin as standards. The gel was silver-stained and theamount of F2L (3 kD) was estimated to 40 ng. G. Biological activity ofF2L (A2, Step 4) on CHO-K1 cells expressing human FPRL2, using theaequorin-based assay.

FIG. 11. Pharmacology of the formyl peptide receptors. A.Concentration-action curves of F2L (⇑), FMLP (▪), WKYMVm (SEQ ID NO:23)(▴), WKYMVM (SEQ ID NO: 21)(▾) and SHAAG (Residues 14-18 of SEQ IDNO: 22)(●) peptides on CHO-K1 cells expressing FPR, FPRL1 or FPRL2,using the aequorin-based assay. Results are expressed as % of theresponse elicited by 20 μM ATP. B. Concentration-action curves of thesame peptides on CHO-K1 cells expressing the three receptors, using acAMP accumulation assay. Results are expressed as % of the cAMP levelobtained in the presence of 10 μM forskolin, but in the absence ofagonists, FSK: forskolin. C. Saturation binding assay (specific binding)on FPRL2-expressing CHO-K1 cells, using F2L bearing a carboxy-terminal[125 I]-Tyr as tracer. D. Competition binding assay on FPRL2-expressingCHO-K1 cells using F2L-[125 I]Tyr as tracer and F2L as competitor. E.and F. Competition binding assay on FPRL1 (E) and FPR (F) -expressingCHO-K1 cells using [125 I]-WKYMVm (SEQ ID NO: 23) as tracer, and WKYMVm(SEQ ID NO: 23)(▴) or F2L (⋄) as competitors. G. Stimulation by F2L ofFPRL2-expressing CHO-K1 cells cultured in the absence or presence of 100ng/ml Pertussis toxin, using the aequorin-based assay. H.Concentration-action curves of acetylated (⋄), non-acetylated (∘) and[7-21]F2L (▭) peptides on FPRL2-expressing CHO-K1 cells using theaequorin assay.

Immunodetection of phosphorylated ERK1/2 in FPRL2-expressing CHO-K1cells following stimulation by F2L for 10 min. J. Kinetics of ERK1/2activation following stimulation by 100 nM F2L. Each experimentdisplayed in A to I was repeated at least three times.

FIG. 12. Expression profile of human FPRL2. A. Transcripts encodinghuman FPRL2 were amplified by RT-PCR in a set of human leukocytepopulations. act.: activated.

Mononucl.: mononuclear cells. iDC: immature dendritic cells. mDC: maturedendritic cells. B. Distribution of FPRL2 in a set of human tissues, byusing quantitative RT-PCR (Taqman). The data were normalized for theexpression of GAPDH used as control. C. Anti-FPRL2 monoclonal antibodieswere characterized by FACS on CHO-K1 cells expressing FPR, FPRL1 andFPRL2. 1C4: bold solid line. 1D2: dotted line. 1E1: dashed line. Controllabeling (IgG2a): thin solid line. The profiles of 1D2 and 1E1 aresuperimposed and cannot therefore be distinguished. D. The expression ofFPRL2 was analyzed by FACS on immature DCs using the three monoclonalantibodies. Anti-FPRL2 Abs: bold solid line. Control labeling (IgG2a):thin solid line. E. Expression of FPRL2 on intact and permeabilized DCsusing 1D2. 1D2: bold solid line. Control labeling (IgG2a): thin solidline. F. Expression of FPRL2 on immature (bold solid line) and mature(thin solid line) DCs using 1D2. Control labeling (IgG2a): dotted lines.

FIG. 13. Biological activity of F2L on primary immune cells. A. and B.Recording of Ca²⁺ flux in monocyte-derived DCs, in response to variousconcentrations of F2L (A) and to 10 nM of FMLP (B). C. Recording of Ca₂₊flux in monocytes, in response to 100 nM and 1 μM of F2L. D. and E.Chemotaxis of monocyte-derived human immature DCs (D) and peripheralblood mononuclear cells (E) in response to F2L. The displayed responsesare representative of four donors, out of five tested.

FIG. 14. Controls of aequorine assays on human FPRL2 expressing cells.(A1-A2: Aequorin medium, A3-A4: ATP 20 μM, A5-A6: Triton 0.1%) (scale:150 000 Relative Light Units (RLU)). Each response graph corresponds tothe indicated position on the FPRL2 96-well plate in Table 2.

FIG. 15. Functional monoclonal antibodies and ligands in aequorineassays on human FPRL2 expressing cells. Scale: 50 000 RLU. Each responsegraph corresponds to the indicated position on the FPRL2 96-well platein Table 2.

DETAILED DESCRIPTION

The invention is based on the discovery that HBP polypeptide is anatural ligand for the orphan G protein coupled receptor FPRL2polypeptide and on methods of using the binding of this ligand to thereceptor in drug screening methods. The known ligand and its interactionwith the receptor FPRL2 polypeptide also provides for the diagnosis ofconditions involving dysregulated receptor activity. The invention alsorelates to a kit comprising FPRL2 polypeptide and homologous sequences,its corresponding polynucleotide and/or recombinant cells expressing thepolynucleotide, to identify agonist, antagonist, inverse agonist andmodulator compounds of the receptor polypeptide and/or its correspondingpolynucleotide. Such kits are useful for the diagnosis, preventionand/or a treatment of diseases and disorders related to FPRL2polypeptide activity.

The invention also relates to novel agonist, antagonist, inverse agonistand modulator compounds of the receptor polypeptide and itscorresponding polynucleotide, identified according to the method of theinvention.

All references referred to below and above are incorporated herein byreference in their entirety.

The invention is based on the finding that a fragment of HBP (HBPpolypeptide) is a natural ligand of the orphan receptor FPRL2 (SEQ IDNO: 2). This invention thus relates to the HBP polypeptideligand/receptor pair, and to functional homologs of the receptor whichalso bind HBP polypeptide and cells transformed by a vector comprisingthe nucleotide sequence encoding the receptor (SEQ ID NO: 1) incombination with the HBP polypeptide ligand. The invention also relatesto a composition consisting essentially of an isolated FPLR2 polypeptideand an isolated HBP polypeptide, as well as to methods of identifyingagents that modulate the activities of FPRL2 polypeptides. The methodsare useful for the identification of agonist, inverse agonist orantagonist compounds useful for the development of new drugs. Theinteraction of FPRL2 with HBP polypeptide is also useful for thedevelopment of diagnostics for diseases related to FPRL2 activity.

The invention encompasses a method of identifying an agent thatmodulates the function of FPLR2, the method comprising: a) contacting aFPLR2 polypeptide with a HBP polypeptide in the presence and absence ofa candidate modulator under conditions permitting the binding of the HBPpolypeptide to the FPLR2 polypeptide; and b) measuring binding of theFPLR2 polypeptide to the HBP polypeptide wherein a decrease in bindingin the presence of the candidate modulator, relative to binding in theabsence of the candidate modulator, identifies the candidate modulatoras an agent that modulates the function of FPLR2 polypeptide.

The invention further encompasses a method of detecting, in a sample,the presence of an agent that modulates the function of FPLR2, themethod comprising: a) contacting a FPLR2 polypeptide with a HBPpolypeptide in the presence and absence of the sample under conditionspermitting the binding of the HBP polypeptide to the FPLR2 polypeptide;and b) measuring binding of the FPLR2 polypeptide to the HBP polypeptidewherein a decrease in binding in the presence of the sample, relative tobinding in the absence of the sample, indicates the presence, in thesample of an agent that modulates the function of FPLR2.

In one embodiment of either of the preceding methods, the measuring isperformed using a method selected from label displacement, surfaceplasmon resonance, fluorescence resonance energy transfer, fluorescencequenching, and fluorescence polarization.

The invention further encompasses a method of identifying an agent thatmodulates the function of FPLR2, the method comprising: a) contacting aFPLR2 polypeptide with a HBP polypeptide in the presence and absence ofa candidate modulator; and b) measuring a signalling activity of theFPLR2 polypeptide, wherein a change in the activity in the presence ofthe candidate modulator relative to the activity in the absence of thecandidate modulator identifies the candidate modulator as an agent thatmodulates the function of FPLR2 polypeptide.

The invention further encompasses a method of identifying an agent thatmodulates the function of FPLR2 polypeptide, the method comprising: a)contacting a FPLR2 polypeptide with a candidate modulator; b) measuringa signalling activity of the FPLR2 polypeptide in the presence of thecandidate modulator; and c) comparing the activity measured in thepresence of the candidate modulator to the activity measured in a samplein which the FPLR2 polypeptide is contacted with a HBP polypeptide atits EC₅₀, wherein the candidate modulator is identified as an agent thatmodulates the function of FPLR2 polypeptide when the amount of theactivity measured in the presence of the candidate modulator is at least20% of the amount induced by the HBP polypeptide present at its EC₅₀.

The invention further encompasses a method of detecting the presence, ina sample, of an agent that modulates the function of FPLR2 polypeptide,the method comprising: a) contacting a FPLR2 polypeptide with HBPpolypeptide in the presence and absence of the sample; b) measuring asignalling activity of the FPLR2 polypeptide; and c) comparing theamount of the activity measured in a reaction containing FPLR2polypeptide and HBP polypeptide without the sample to the amount of theactivity measured in a reaction containing FPLR2 polypeptide, HBPpolypeptide and the sample, wherein a change in the activity in thepresence of the sample relative to the activity in the absence of thesample indicates the presence, in the sample, of an agent that modulatesthe function of FPLR2 polypeptide.

The invention further encompasses a method of detecting the presence, ina sample, of an agent that modulates the function of FPLR2 polypeptide,the method comprising: a) contacting a FPLR2 polypeptide with thesample; b) measuring a signalling activity of the FPLR2 polypeptide inthe presence of the sample; and c) comparing the activity measured inthe presence of the sample to the activity measured in a reaction inwhich the FPLR2 polypeptide is contacted with a HBP polypeptide presentat its EC₅₀, wherein an agent that modulates the function of FPLR2polypeptide is detected if the amount of the activity measured in thepresence of the sample is at least 20% of the amount induced by the HBPpolypeptide present at its EC₅₀.

In one embodiment of each of the preceding methods, the HBP polypeptideis detectably labeled. In a preferred embodiment, the HBP polypeptide isdetectably labeled with a moiety selected from the group consisting of aradioisotope, a fluorophore, a quencher of fluorescence, an enzyme, andan affinity tag.

In an embodiment of each of the preceding methods, the contacting isperformed in or on a cell expressing the FPLR2 polypeptide.

In an embodiment of each of the preceding methods the contacting isperformed in or on synthetic liposomes.

In an embodiment of each of the preceding methods the contacting isperformed in or on virus-induced budding membranes containing a FPLR2polypeptide.

In an embodiment of each of the preceding methods the contacting isperformed using a membrane fraction from cells expressing the FPLR2polypeptide.

In an embodiment of each of the preceding methods the measuring isperformed using a method selected from the group consisting of labeldisplacement, surface plasmon resonance, fluorescence resonance energytransfer, fluorescence quenching, and fluorescence polarization.

In an embodiment of each of the preceding methods the agent is selectedfrom the group consisting of a natural or synthetic peptide orpolypeptide, an antibody or antigen-binding fragment thereof, a lipid, acarbohydrate, a nucleic acid, an antisense nucleotide, and a smallorganic molecule.

In one embodiment of the methods wherein a signalling activity ismeasured, the step of measuring a signalling activity of the FPLR2polypeptide comprises detecting a change in the level of a secondmessenger.

In another embodiment of the methods wherein a signalling activity ismeasured, the step of measuring a signalling activity comprisesmeasurement of guanine nucleotide binding or exchange, adenylate cyclaseactivity, cAMP, Protein Kinase C activity, phosphatidylinositolbreakdown, diacylglycerol, inositol trisphosphate, intracellularcalcium, arachinoid acid, MAP kinase activity, tyrosine kinase activity,or reporter gene expression.

In one embodiment, the step of measuring a signalling activity comprisesusing an aequorin-based assay.

The invention further comprises a method of modulating the activity of aFPLR2 polypeptide in a cell, the method comprising the step ofdelivering to the cell an agent that modulates the activity of a FPLR2polypeptide, such that the activity of FPLR2 polypeptide is modulated.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of FPLR2 polypeptide signalling,the method comprising: a) contacting a tissue sample with an antibodyspecific for a FPLR2 polypeptide; b) detecting binding of the antibodyto the tissue sample; and c) comparing the binding detected in step (b)with a standard, wherein a difference in binding relative to thestandard is diagnostic of a disease or disorder characterized bydysregulation of FPLR2 polypeptide.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of FPLR2 polypeptide signalling,the method comprising: a) isolating nucleic acid from a tissue sample;b) amplifying a FPLR2 polynucleotide, using the nucleic acid as atemplate; and c) comparing the amount of amplified FPLR2 polynucleotideproduced in step (b) with a standard, wherein a difference in the amountof amplified FPLR2 polynucleotide relative to the standard is diagnosticof a disease or disorder characterized by dysregulation of FPLR2polypeptide.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of FPLR2 polypeptide signalling,the method comprising: a) isolating nucleic acid from a tissue sample;b) amplifying a FPLR2 polynucleotide, using the nucleic acid as atemplate; and c) comparing the sequence of the amplified FPLR2polynucleotide produced in step (b) with a standard, wherein adifference in the sequence, relative to the standard is diagnostic of adisease or disorder characterized by dysregulation of FPLR2 polypeptide.In one embodiment, the step of amplifying comprises RT/PCR. In anotherembodiment, the standard is SEQ ID NO: 1. In another embodiment, thestep of comparing the sequence comprises minisequencing. In anotherembodiment, the step of comparing the amount is performed using amicroarray.

The invention further encompasses a composition comprising or consistingessentially of an isolated FPLR2 polypeptide and an isolated HBPpolypeptide. An isolated FPLR2 polypeptide and an isolated HBPpolypeptide together can form a complex that is useful for theidentification of agents that modulate their interaction, theidentification of agents that modulate the activity of FPLR2polypeptides, and the identification of individuals suffering from adisease or disorder mediated by or involving FPLR2 polypeptide.Complexed or uncomplexed (i.e., bound or unbound) isolated FPLR2polypeptide and isolated HBP polypeptide is thus the essential elementor basis of the assays and methods of the invention. The composition“consisting essentially of” an isolated FPLR2 polypeptide and anisolated HBP polypeptide can comprise additional components, however,such additional components are not essential to the novel interactionupon which the invention is based. The composition “consistingessentially of” an isolated FPLR2 polypeptide and an isolated HBPpolypeptide is distinct from and excludes naturally occurring complexesbetween FPLR2 polypeptides and HBP polypeptide, present e.g., in cells,tissues or in cell or tissue extracts. The composition of the inventionis also distinct from and excludes complexes between FPLR2 polypeptidesexpressed from recombinant constructs and naturally-occurring HBPpolypeptide.

Kits according to the invention are useful, for example, for screeningfor agents that modulate the activity of FPLR2 polypeptide, identifyingthe presence of an agent that modulates FPLR2 polypeptide in a sample,or for diagnosis of a disease or disorder characterized by dysregulationof FPLR2 polypeptide. Kits according to the invention will additionallycomprise packaging materials necessary for such kits. Kits according tothe invention can additionally comprise a standard. In one embodiment,the standard is a sample from an individual not affected by a disease ordisorder characterized by dysregulation of FPLR2 polypeptide.

As used herein, the term “formyl peptide receptor-like 2 (FPRL2)polypeptide” refers to a polypeptide having at least 80% amino acididentity, preferably 85%, 90%, 95%, or higher, up to and including 100%identity, with SEQ ID NO: 2, and which has FPRL2 activity i.e., theFPRL2 polypeptide binds a HBP polypeptide or a functional fragmentthereof. An FPRL2 polypeptide may also be a functional fragment of SEQID NO: 2 i.e. a portion of SEQ ID NO:2 which is still capable of bindingto a HBP polypeptide or a functional fragment thereof. A functionalfragment of SEQ ID NO: 2 may comprise at least 10, 20, 30, 40, 50, 60,70, 80, 90, or 95% of the amino acids of the sequence represented by SEQID NO:2.

Optimally, a FPRL2 polypeptide also has FPRL2 signalling activity asdefined herein.

As used herein, “FPRL2 polypeptide activity” refers to specific bindingto or signalling by a HBP polypeptide as defined herein.

A homologous sequence (which may exist in other mammal species orspecific groups of human populations), where homology indicates sequenceidentity, means a sequence which presents a high sequence identity (morethan 80%, 85%, 90%, 95% or 98% sequence identity) with the completehuman nucleotide of SEQ ID NO: 1 or the complete human amino acidsequence of SEQ ID NO: 2. A functional homolog is characterized by theability to bind a HBP polypeptide as defined herein or by the ability toinitiate or propagate a signal in response to ligand binding, or both.

Homologous sequences of a sequence according to the invention mayinclude an amino acid or nucleotide sequence encoding a similar receptorwhich exists in other animal species (rat, mouse, cat, dog, etc.) or inspecific human population groups, but which are involved in the samebiochemical pathway.

Such homologous sequences may comprise additions, deletions orsubstitutions of one or more amino acids or nucleotides, which do notsubstantially alter the functional characteristics of the receptoraccording to the invention. That is, homologs will have at least 90% ofthe activity of wt full length human FPRL2 polypeptide and will bind HPBpolypeptide specifically.

Such homologous sequences can also be nucleotide sequences of more than50, 100, 200, 300, 400, 600, 800 or 1000 nucleotides which are able tohybridize to the complete human FPRL2 sequence under stringenthybridisation conditions (such as the ones described by SAMBROOK et al.,Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor Laboratorypress, New York). An example of “stringent hybridization conditions” isas follows: hybridize in 50% formamide, 5×SSC, 50 mM sodium phosphate(pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, 50 μg/mlsonicated salmon sperm DNA, 0.1% SDS and 10% dextran sulfate at 42° C.;and wash at 42° C. (or higher, e.g., up to two degrees C. below theT_(m) of the perfect complement of the probe sequence) in 0.2×SSC and0.1% SDS.

As used herein, the term “formyl peptide receptor-like 2 (FPRL2)signalling activity” refers to the initiation or propagation ofsignalling by a FPRL2 polypeptide. FPRL2 signalling activity ismonitored by measuring a detectable step in a signalling cascade byassaying one or more of the following: stimulation of GDP for GTPexchange on a G protein; alteration of adenylate cyclase activity,protein kinase C modulation; phosphatidylinositol breakdown (generatingsecond messengers diacylglycerol, and inositol trisphosphate);intracellular calcium flux; activation of MAP kinases; modulation oftyrosine kinases; or modulation of gene or reporter gene activity. Adetectable step in a signalling cascade is considered initiated ormediated if the measurable activity is altered by 10% or more above orbelow a baseline established in the substantial absence of a HBPpolypeptide relative to any of the FPRL2 polypeptide activity assaysdescribed herein below. The measurable activity can be measureddirectly, as in, for example, measurement of cAMP or diacylglycerollevels. Alternatively, the measurable activity can be measuredindirectly, as in, for example, a reporter gene assay.

The term “a heme binding protein (HBP) polypeptide” refers to apolypeptide having at least 50% or higher identity to SEQ ID NO: 18, andthe defined polypeptide specifically binds to and activates a signalingactivity of a FPRL2 polypeptide. Preferrably, the polypeptide is atleast 55%, or higher identity to SEQ ID NO: 18. Preferrably, thepolypeptide is at least 60%, or 70%, or 80%, 85%, 90%, 95%, or 98% orhigher identity to SEQ ID NO: 18.

A HBP polypeptide may also be a functional fragment of SEQ ID NO: 18i.e. a portion of SEQ ID NO: 18 which is still capable of binding to aFPRL2 polypeptide or a functional fragment thereof. A functionalfragment of SEQ ID NO: 18 comprise at least 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21, or a number in the range betweenany two of the aforementioned numbers of amino acids of the sequencerepresented by SEQ ID NO: 18.

The term “specifically binds” means that the HBP polypeptide has anEC₅₀, IC₅₀, or a K_(d) of 100nM or less. “HBP polypeptide” also refersto a fragment of a polypeptide meeting the preceding definition, whereinthe fragment retains at least 50% of the binding activity and level ofsignaling activation of the full length polypeptide of SEQ ID NO: 18. AHBP polypeptide also includes a analog, variant or some shortpolypeptide from COOH-terminal end and/or NH2-terminal end of SEQ ID NO18. A HBP polypeptide can also comprise chemical and/or amino acidadditions, insertions, deletions or substitutions relative to SEQ ID NO:18, as long as the resulting polypeptide retains at least 50% of thebinding activity and level of signaling activation of the full lengthpolypeptide represented by SEQ ID NO: 18. A HBP polypeptide, cancomprise additional sequences, as in for example, a HBP fusion protein.Non-limiting examples of fusion partners includeglutathione-S-transferase (GST), maltose binding protein, alkalinephosphatase, thioredoxin, green fluorescent protein (GFP), histidinetags (e.g., 6X or greater His (SEQ ID NO: 36)), or epitope tags (e.g.,Myc tag, FLAG tag). An HBP polypeptide can be a polypeptide sequencerepresented by SEQ ID NO: 18, with or without an acetyl group at theN-terminus. An HBP polypeptide can be a polypeptide sequence representedby SEQ ID NO: 18, with or without a label such as biotin or any otherdye (fluorescent dye) or with a radioisotope. Where a label is present,it may attach, for example, through an acetyl group at the N-terminus ofthe HBP polypeptide. One or more combinations of the above features arewithin the scope of the invention.

Homologous sequences of SEQ ID NO: 18 according to the invention mayinclude an amino acid or nucleotide sequence encoding a similar sequencewhich exists in other animal species (rat, mouse, human, cat, dog, etc.)or in specific human population groups, but which are involved in thesame biochemical pathway.

Such homologous sequences may comprise additions, deletions orsubstitutions of one or more amino acids or nucleotides, which do notsubstantially alter the functional characteristics of the peptideaccording to the invention. That is, homologs will have at least 90% ofthe activity of an amino acid sequence represented by SEQ ID NO: 18 andwill bind or activate FPRL2 specifically.

As used herein, the term “detectable step” refers to a step that can bemeasured, either directly, e.g., by measurement of a second messenger ordetection of a modified (e.g., phosphorylated) protein, or indirectly,e.g., by monitoring a downstream effect of that step. For example,adenylate cyclase activation results in the generation of cAMP. Theactivity of adenylate cyclase can be measured directly, e.g., by anassay that monitors the production of cAMP in the assay, or indirectly,by measurement of actual levels of cAMP.

Preferably, a recombinant cell according to the invention is arecombinant cell transformed by a plasmid, cosmid or viral vector,preferably a baculovirus, an adenovirus, or a semliki forest virus, andthe cell is preferably selected from the group consisting of bacterialcells, yeast cells, insect cells or mammal cells.

According to a preferred embodiment of the present invention, the cellis selected from the group consisting of COS-7 cells, a CHO cell, a LM(TK-) cell, a NIH-3T3 cell, HEK-293 cell, K-562 cell or a 1321N1astrocytoma cell. Other transfectable cell lines are also useful,however. Preferably, the vector comprises regulatory elementsoperatively linked to the polynucleotide sequence encoding the receptoraccording to the invention, so as to permit expression thereof.

Another aspect of the present invention is related to the use of aspecific active portion of the sequences. As used herein, an “activeportion” refers to a portion of a sequence that is of sufficient size toexhibit normal or near normal pharmacology (e.g., receptor activity (asdefined herein), the response to an activator or inhibitor, or ligandbinding are at least 90% of the level of activity, response, or bindingexhibited by a wild type receptor). “A portion” as it refers to asequence encoding a receptor, refers to less than 100% of the sequence(i.e., 99, 90, 80, 70, 60, 50% etc. . . . ). The active portion could bea receptor which comprises a partial deletion of the complete nucleotideor amino acid sequence and which still maintains the active site(s) andprotein domain(s) necessary for the binding of and interaction with aspecific ligand, preferably HBP polypeptide.

In another embodiment of any of the preceding methods, the contacting isperformed in or on synthetic liposomes (Mirzabekov et al., 2000) orvirus-induced budding membranes containing a FPRL2 polypeptide. (seePatent application WO0102551, Virus-like particles, their Preparationand their Use preferably in Pharmaceutical Screening and FunctionalGenomics (2001) incorporated herein by reference).

As used herein, “ligand” refers to a moiety that is capable ofassociating or binding to a receptor. According to the method of theinvention, a ligand and a receptor have a binding constant that issufficiently strong to allow detection of binding by an assay methodthat is appropriate for detection of a ligand binding to a receptor(e.g. a second messenger assay to detect an increase or decrease in theproduction of a second messenger in response to ligand binding to thereceptor, a binding assay to measure protein-ligand binding or animmunoassay to measure antibody-antigen interactions). A ligandaccording to the invention includes the actual molecule that binds areceptor or a ligand may be any nucleotide, antibody, antigen, enzyme,small organic molecule, peptide, polypeptide or nucleic acid capable ofbinding to the receptor. A ligand is preferably a HBP polypeptide, apeptide or a nucleic acid sequence. According to the method of theinvention, a ligand and receptor specifically bind to each other (e.g.via covalent or hydrogen bonding or via an interaction between, forexample, a protein and a ligand, an antibody and an antigen or proteinsubunits).

Another aspect of the present invention is related to a method for thescreening, detection and recovery of candidate modulators of a receptorof the invention comprising the steps of: contacting a cell expressingFPRL2 polypeptide with HBP polypeptide under conditions which permitbinding of HBP polypeptide to FPRL2 polypeptide, in the presence of thecandidate modulator, performing a second messenger assay, and comparingthe results of the second messenger assay obtained in the presence orabsence of the candidate modulator.

Another aspect of the present invention is related to a method for thescreening, detection and possible recovery of candidate modulators of areceptor of the invention comprising the steps of: contacting a cellmembrane expressing FPRL2 polypeptide with HBP polypeptide underconditions which permit binding of HBP polypeptide to FPRL2 polypeptide,performing a second messenger assay, and comparing the results of thesecond messenger assay obtained in the presence or absence of thecandidate modulator.

In another embodiment, the step of measuring a signalling activity ofthe FPRL2 polypeptide comprises detecting a change in the level of asecond messenger.

A further aspect of the present invention is related to the unknownagonist and/or antagonist compounds identified and/or recovered by themethod of the invention, as well as to a diagnostic kit comprising the(unknown) compounds or a pharmaceutical composition (including avaccine) comprising an adequate pharmaceutical carrier and a sufficientamount of the (unknown) compound.

An antagonist compound according to the invention means a molecule or agroup of molecules able to bind to the receptor according to theinvention and block the binding of natural compounds (HBP polypeptide).

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of FPRL2 polypeptide signalling,the method comprising: a) contacting a tissue sample with an antibodyspecific for a FPRL2 polypeptide and an antibody specific for a FPRL2ligand; b) detecting binding of the antibodies to the tissue sample; andc) comparing the binding detected in step (b) with a standard, wherein adifference in binding of either antibody or both, relative to thestandard, is diagnostic of a disease or disorder characterized bydysregulation of FPRL2 polypeptide.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of FPRL2 polypeptide signalling,the method comprising: a) isolating a tissue sample; b) measuring theconcentration of HBP polypeptide; and c) comparing the amount of LIGANDmeasured in step (b) with a standard, wherein a difference in the amountof HBP polypeptide relative to the standard is diagnostic of a diseaseor disorder characterized by dysregulation of FPRL2 polypeptide.

A further aspect of the present invention is related to a non-humanmammal comprising a homozygous null mutation (homozygous “knock-out”) ofthe polynucleotide sequence encoding the FPRL2 polypeptide receptoraccording to the invention, or a transgenic non-human mammal that overexpresses a FPRL2 polypeptide above the natural level of expression. Asused herein. “above the natural level of expression” refers to a levelthat is at least 2-fold, preferably 5-fold, more preferably 10-fold andmost preferably 100-fold or more (i.e., 150-fold, 200-fold, 250-fold,500-fold, 1000-fold, 10,000-fold etc.) as compared to the level ofexpression of the endogenous receptor in its normal native context. Atransgenic non-human mammal according to the invention will express thetransgene in at least one tissue or cell type but can express the FPRL2polypeptide transgene in all tissues and cells. A transgenic non-humanmammal can be obtained by a method well known by a person skilled in theart, for instance, as described in document WO 98/20112 using theclassical technique based upon the transfection of embryonic stem cells,preferably according to the method described by Carmeliet et al.(Nature, Vol. 380, p. 435-439, 1996).

“Gene targeting” is a type of homologous recombination that occurs whena fragment of genomic DNA is introduced into a mammalian cell and thatfragment locates and recombines with endogenous homologous sequences asexemplified in U.S. Pat. No. 5,464,764, and U.S. Pat. No. 5,777,195, thecontents of which are hereby incorporated by reference herein in theirentireties. As used herein the term “transgenic animal” refers to anon-human animal in which one or more, and preferably essentially all,of the cells of the animal contain a transgene introduced by way ofhuman intervention, such as by transgenic techniques known in the art.The transgene can be introduced into the cell, directly or indirectly byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus.

Preferably, the transgenic non-human mammal overexpressing thepolynucleotide encoding the FPRL2 polypeptide receptor according to theinvention comprises the polynucleotide incorporated in a DNA constructwith an inducible promoter allowing the overexpression of the receptorand possibly also tissue and cell-specific regulatory elements.

In one embodiment, the kits according to the invention comprise reagentsfor measuring the binding of a HBP polypeptide to a FPRL2 polypeptide.In another embodiment, the kit comprises reagents for measuring asignalling activity of a FPRL2 polypeptide.

In one embodiment, a screening or diagnostic kit according to theinvention includes a FPRL2 receptor polypeptide or a cellular membranepreparation comprising a FPRL2 polypeptide and one or more HBPpolypeptide in separate containers. Such kits can additionally compriseall the necessary means and media for performing a detection of specificbinding (for example of HBP polypeptide) to the FPRL2 polypeptidereceptor according to the invention. Binding or signalling activity canbe correlated with a method of monitoring one or more of the symptoms ofthe diseases described hereafter.

The diagnostic kits can thus further comprise elements necessary for aspecific diagnostic measurement, or, for example, the measurements ofbound compounds using high throughput screening techniques known to theperson skilled in the art, e.g., the techniques described in WO00/02045. Such kits can be used, e.g. to monitor dosage andeffectiveness of FPRL2 polypeptide modulating agents used for treatment.The high throughput screening diagnostic dosage and monitoring can beperformed by using various solid supports, such as microtiter plates orbiochips selected by the person skilled in the art.

In a pharmaceutical composition according to the invention, the adequatepharmaceutical carrier is a carrier of solid, liquid or gaseous form,which can be selected by the person skilled in the art according to thetype of administration and the possible side effects of the compoundadministered to modulate FPRL2 polypeptide activity. The pharmaceuticalcarrier useful according to the invention does not include tissueculture medium or other media comprising serum. The ratio between thepharmaceutical carrier and the specific compound can be selected by theperson skilled in the art according to the patient treated, theadministration and the possible side effects of the compound, as well asthe type of disease of disorder treated or sought to be prevented.

The pharmaceutical composition finds advantageous applications in thefield of treatment and/or prevention of various diseases or disorders,preferably selected from the group consisting of cell migration, cancer,development of tumours and tumour metastasis, inflammatory andneoplastic processes, wound and bone healing and dysfunction ofregulatory growth functions, obesity, anorexia, bulimia, acute heartfailure, hypotension, hypertension, urinary retention, osteoporosis,angina pectoris, restenosis, atherosclerosis, thrombosis and othercardiovascular diseases, autoimmune and, diseases characterized byexcessive smooth muscle cell proliferation, aneurysms, diseasescharacterized by loss of smooth muscle cells or reduced smooth musclecell proliferation, stroke, ischemia, ulcers, allergies, prostatichypertrophy, migraine, vomiting, psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, depression,delirium, dementia and severe mental retardation, degenerative diseases,neurodegenerative diseases such as ALzheimer's disease or Parkinson'sdisease, and dyskinasias, such as Huntington's disease or Gilles de laTourett's syndrome and other related diseases including thrombosis andother cardiovascular diseases, autoimmune and inflammatory diseases suchas psoriasis, Eczeme, inflammatory and trophic diseases of skin,rheumatoid arthritis, scleroderma, lupus, polymyositis, dermatomysitis,Crohn's disease, inflammatory bowel disease (IBD), Irritable BowelSyndrome, Ulcerative Colitis, Asthma, Chronic Obstructive PulmonaryDisease, Allergic Rhinitis, Fibromyalgia, Organ Transplant Rejection,Graft versus host disease, Multiple Sclerosis, Acute, Ischemic Stroke,Infectious diseases, Hepatitis A, Hepatitis B, Hepatitis C, Sepsis,Septic shock, Chronic bronchitis, infections such as bacterial, fungal,protozoan and viral infections, such as infections caused by HIV1 andHIV2, and pain, cancer, anorexia, bulimia, asthma, acute heart failure,hypertension, urinary retention, osteoporosis, angina pectoris,myocardial infarction, ulcers, allergies, benign prostatic hypertrophy,and Type 1 Diabetes, Type 2 Diabetes, Osteoarthritis, DiabeticRetinopathy, Diabetic Nephropathy and fertility dysfunctions, foetaldevelopmental disorders

Among the mentioned diseases the preferred applications are related totherapeutic agents targeting 7TM receptors that can play a function inpreventing, improving or correcting dysfunctions or diseases, including,but not limited to cell migration, cancer, development of tumours andtumour metastasis, inflammatory and neoplastic processes, wound and bonehealing and dysfunction of regulatory growth functions, obesity,anorexia, bulimia, acute heart failure, hypotension, hypertension,urinary retention, osteoporosis, angina pectoris, restenosis,atherosclerosis, thrombosis and other cardiovascular diseases,autoimmune and, diseases characterized by excessive smooth muscle cellproliferation, aneurysms, diseases characterized by loss of smoothmuscle cells or reduced smooth muscle cell proliferation, stroke,ischemia, ulcers, allergies, prostatic hypertrophy, migraine, vomiting,psychotic and neurological disorders, including anxiety, schizophrenia,manic depression, depression, delirium, dementia and severe mentalretardation, degenerative diseases, neurodegenerative diseases such asAlzheimer's disease or Parkinson's disease, and dyskinasias, such asHuntington's disease or Gilles de la Tourett's syndrome and otherrelated diseases including thrombosis and other cardiovascular diseases,autoimmune and inflammatory diseases such as psoriasis, Eczeme,inflammatory and trophic diseases of skin, rheumatoid arthritis,scleroderma, lupus, polymyositis, dermatomysitis, Crohn's disease,inflammatory bowel disease (IBD), Irritable Bowel Syndrome, UlcerativeColitis, Asthma, Chronic Obstructive Pulmonary Disease, AllergicRhinitis, Fibromyalgia, Organ Transplant Rejection, Graft versus hostdisease, Multiple Sclerosis, Acute, Ischemic Stroke, Infectiousdiseases, Hepatitis A, Hepatitis B, Hepatitis C, Sepsis, Septic shock,Chronic bronchitis, infections such as bacterial, fungal, protozoan andviral infections, such as infections caused by HIV1 and HIV2, and pain,cancer, anorexia, bulimia, asthma, acute heart failure, hypertension,urinary retention, osteoporosis, angina pectoris, myocardial infarction,ulcers, allergies, benign prostatic hypertrophy, and Type 1 Diabetes,Type 2 Diabetes, Osteoarthritis, Diabetic Retinopathy, DiabeticNephropathy and fertility dysfunctions, foetal developmental disorders.

The invention further encompasses an agent which modulates FPRL2polypeptide activity identified by the method or detected in a sample asmentioned above.

The invention further encompasses the use of said agent for themodulation of FPRL2 polypeptide activity.

The invention further encompasses the use of said agent for themanufacture of a medicament for the treatment of FPRL2polypeptide-related diseases or for the manufacture of a kit for themodulation of FPRL2 polypeptide activity.

The invention further encompasses a pharmaceutical compositioncomprising an adequate pharmaceutical carrier or diluent and asufficient amount of said agent.

The invention further encompasses a pharmaceutical composition accordingto according to the above-mentioned, further comprising a vesicle or anadjuvant able to modulate the immune response of a patient to which itis administered.

The invention further encompasses the use of the above-mentionedpharmaceutical composition for the manufacture of a medicament for thetreatment of FPRL2 polypeptide—related diseases or for the manufactureof a kit for the modulation of FPRL2 polypeptide.

The invention also relates to the use of a HBP polypeptide for themodulation of FPRL2 polypeptide activity in vivo and/or in vitro.

The invention also relates to the use of a HBP polypeptide in thevalidation of a non-human mammal comprising a partial or total deletionof the polynucleotide encoding FPRL2 polypeptide.

The invention also relates to the use of a HBP polypeptide in thevalidation of a non-human mammal overexpressing the polynucleotideencoding FPRL2 polypeptide.

As used herein, an “antagonist” is a ligand which competitively binds toa receptor at the same site as an agonist, but does not activate anintracellular response initiated by an active form of the receptor. Anantagonist thereby inhibits the intracellular response induced by anagonist, for example HBP polypeptide, by at least 10%, preferably15-25%, more preferably 25-50% and most preferably, 50-100%, as comparedto the intracellular response in the presence of an agonist and in theabsence of an antagonist.

As used herein, an “agonist” refers to a ligand that activates anintracellular response when it binds to a receptor at concentrationsequal to or lower than HBP polypeptide concentrations which induce anintracellular response. An agonist according to the invention canincrease the intracellular response mediated by a receptor by at least2-fold, preferably 5-fold, more preferably 10-fold and most preferably100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold,1000-fold, 10,000-fold etc . . . ), as compared to the intracellularresponse in the absence of agonist. An agonist according to theinvention may promotes internalization of a cell surface receptor suchthat the cell surface expression of a receptor is increased by at least2-fold, preferably 5-fold, more preferably 10-fold and most preferably,100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold,1000-fold, 10,000-fold etc . . . ), as compared to the number of cellsurface receptors present on the surface of a cell in the absence of anagonist. In another embodiment of the invention, an agonist stabilizes acell surface receptor and increases the cell surface expression of areceptor by at least 2-fold, preferably 5-fold, more preferably 10-foldand most preferably, 100-fold or more (i.e., 200-fold, 250-fold,500-fold, 1000-fold, 10,000-fold etc . . . ), as compared to the numberof cell surface receptors present on the surface of a cell in theabsence of agonist.

As used herein, an “inverse agonist” refers to a ligand which decreasesa constitutive activity of a cell surface receptor when it binds to areceptor. An inverse agonist according to the invention can decrease theconstitutive intracellular response mediated by a receptor by at least2-fold, preferably 5-fold, more preferably 10-fold and most preferably100-fold or more (i.e., 150-fold, 200-fold, 250-fold, 500-fold,1000-fold, 10,000-fold etc . . . ), as compared to the intracellularresponse in the absence of inverse agonist.

An “inhibitor” compound according to the invention is a moleculedirected against the receptor or against the natural ligand for thereceptor that decreases the binding of the ligand to the receptor by atleast 10%, preferably 15-25%, more preferably 25-50% and mostpreferably, 50-100%, in the presence of HBP polypeptide, as compared tothe binding in the presence of HBP polypeptide and in the absence ofinhibitor. An “inhibitor” compound of the invention can decrease theintracellular response induced by an agonist, for example HBPpolypeptide, by at least 10%, preferably 15-25%, more preferably 25-50%and most preferably, 50-100%. An “inhibitor” also refers to a nucleotidesequence encoding an inhibitor compound of the invention. An inhibitor,useful according to the present invention, includes, but is not limitedto an antibody which specifically binds to at least a portion of FPRL2polypeptide which is required for signal transduction through FPRL2polypeptide (such as the ligand binding site), or chemical compoundswhich are capable of blocking or reducing (e.g., by at least 10%) thesignal transduction pathway which is coupled to the FPRL2 polypeptidereceptor. Such inhibitors include, but are not limited to sub-lethaldoses of pertussis toxin, N-ethylmaleimide (NEM; Sigma), dibutyryl cAMP(Boehringer Mannheim, Corp.), and H-89(N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide-HCL;Calbiochem).

As used herein, “natural ligand” refers to a naturally occurring ligand,found in nature, which binds to a receptor in a manner that is at leastequivalent to HBP polypeptide. A “natural ligand” does not refer to anengineered ligand that is not found in nature and that is engineered tobind to a receptor, where it did not formerly do so in a mannerdifferent, either in degree or kind, from that which it was engineeredto do. Such an engineered ligand is no longer naturally-occurring but is“non-natural” and is derived from a naturally occurring molecule.

As used herein, a “modulator” refers to a compound that increases ordecreases the cell surface expression of a receptor of the invention,increases or decreases the binding of a ligand to a receptor of theinvention, or any compound that increases or decreases the intracellularresponse initiated by an active form of the receptor of the invention,either in the presence or absence or an agonist, and in the presence ofa ligand for the receptor, for example HBP polypeptide. A modulatorincludes an agonist, antagonist, inhibitor or inverse agonist, asdefined herein. A modulator can be for example, a polypeptide, apeptide, an antibody or antigen-binding fragment thereof, a lipid, acarbohydrate, a nucleic acid, and a small organic molecule. Candidatemodulators can be natural or synthetic compounds, including, forexample, synthetic small molecules, compounds contained in extracts ofanimal, plant, bacterial or fungal cells, as well as conditioned mediumfrom such cells.

As used herein, “increase” and “decrease” refer to a change in ligandbinding to the FPRL2 polypeptide receptor and/or cell signalling throughFPRL2 polypeptide of at least 10%. An “increase” or “decrease” inbinding or signalling is preferably measured in response to contactingFPRL2 polypeptide with a ligand in the presence of a candidatemodulator, wherein the change in binding or signalling is relative tothe binding or signalling in the absence of the candidate modulator.

As used herein, the term “small molecule” refers to a compound havingmolecular mass of less than 3000 Daltons, preferably less than 2000 or1500, still more preferably less than 1000, and most preferably lessthan 600 Daltons. A “small organic molecule” is a small molecule thatcomprises carbon.

As used herein, the terms “change”, “difference”, “decrease”, or“increase” as applied to e.g., binding or signalling activity or amountof a substance refer to an at least 10% increase or decrease in binding,signalling activity, or for example, level of mRNA, polypeptide orligand relative to a standard in a given assay.

As used herein, the term “dysregulation” refers to the signallingactivity of FPRL2 polypeptide in a sample wherein:

a) a 10% or greater increase or decrease in the amount of one or more ofFPRL2 polypeptide, ligand or mRNA level is measured relative to astandard, as defined herein, in a given assay or;

b) at least a single base pair change in the FPRL2 polypeptide codingsequence is detected relative to SEQ ID NO: 1, and results in analteration of FPRL2 polypeptide ligand binding or signalling activity asdefined in paragraphs a), c) or d) or;

c) a 10% or greater increase or decrease in the amount of FPRL2polypeptide ligand binding activity is measured relative to a standard,as defined herein, in a given assay or;

d) a 10% or greater increase or decrease in a second messenger, asdefined herein, is measured relative to the standard, as defined herein,in a given assay.

As used herein, the term “conditions permitting the binding of HBPpolypeptide to a FPRL2 polypeptide” refers to conditions of, forexample, temperature, salt concentration, pH and protein concentrationunder which FPRL2, binds FPRL2 polypeptide. Exact binding conditionswill vary depending upon the nature of the assay, for example, whetherthe assay uses viable cells or only a membrane fraction of cells.However, because FPRL2 polypeptide is a cell surface protein favoredconditions will generally include physiological salt (90 mM) and pH(about 7.0 to 8.0). Temperatures for binding can vary from 15° C. to 37°C., but will preferably be between room temperature and about 30° C. Theconcentration of HBP polypeptide in a binding reaction will also vary,but will preferably be about 10 pM to 10 nM (e.g., in a reaction withradiolabelled tracer HBP polypeptide).

As used herein, the term “sample” refers to the source of moleculesbeing tested for the presence of an agent or modulator compound thatmodulates binding to or signalling activity of a FPRL2 polypeptide. Asample can be an environmental sample, a natural extract of animal,plant yeast or bacterial cells or tissues, a clinical sample, asynthetic sample, or a conditioned medium from recombinant cells or afermentation process. The term “tissue sample” refers to a tissue thatis tested for the presence, abundance, quality or an activity of a FPRL2polypeptide, a nucleic acid encoding a FPRL2 polypeptide, a FPRL2 ligandor an agent or compound that modifies the ligand binding or activity ofa FPRL2 polypeptide.

As used herein, a “tissue” is an aggregate of cells that perform aparticular function in an organism. The term “tissue” as used hereinrefers to cellular material from a particular physiological region. Thecells in a particular tissue can comprise several different cell types.A non-limiting example of this would be brain tissue that furthercomprises neurons and glial cells, as well as capillary endothelialcells and blood cells, all contained in a given tissue section orsample. In addition to solid tissues, the term “tissue” is also intendedto encompass non-solid tissues, such as blood.

As used herein, the term “membrane fraction” refers to a preparation ofcellular lipid membranes comprising a FPRL2 polypeptide. As the term isused herein, a “membrane fraction” is distinct from a cellularhomogenate, in that at least a portion (i.e., at least 10%, andpreferably more) of non-membrane-associated cellular constituents hasbeen removed. The term “membrane associated” refers to those cellularconstituents that are either integrated into a lipid membrane or arephysically associated with a component that is integrated into a lipidmembrane.

As used herein, the “second messenger assay” preferably comprises themeasurement of guanine nucleotide binding or exchange, adenylatecyclase, intra-cellular cAMP, intracellular inositol phosphate,intra-cellular diacylglycerol concentration, arachidonic acidconcentration, MAP kinase(s) or tyrosine kinase(s), protein kinase Cactivity, or reporter gene expression or an aequorin-based assayaccording to methods known in the art and defined herein.

As used herein, the term “second messenger” refers to a molecule,generated or caused to vary in concentration by the activation of aG-Protein Coupled Receptor, that participates in the transduction of asignal from that GPCR. Non-limiting examples of second messengersinclude cAMP, diacylglycerol, inositol trisphosphate, arachidonic acidrelease, and intracellular calcium. The term “change in the level of asecond messenger” refers to an increase or decrease of at least 10% inthe detected level of a given second messenger relative to the amountdetected in an assay performed in the absence of a candidate modulator.

As used herein, the term “aequorin-based assay” refers to an assay forGPCR activity that measures intracellular calcium flux induced byactivated GPCRs, wherein intracellular calcium flux is measured by theluminescence of aequorin expressed in the cell.

As used herein, the term “binding” refers to the physical association ofa ligand (e.g., a ligand such as HBP polypeptide, or an antibody) with areceptor (e.g., FPRL2). As the term is used herein, binding is“specific” if it occurs with an EC₅₀ or a K_(d) of 1 mM less, generallyin the range of 100 nM to 10 pM For example, binding is specific if theEC₅₀ or K_(d) is 100 nM, 50 nM, 10 nM, 1 nM, 950 pM, 900 pM, 850 pM, 800pM, 750 pM, 700 pM, 650 pM, 600 pM, 550 pM, 500 pM, 450 pM, 350 pM, 300pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 25 pM, 10 pM or less.

As used herein, the term “EC₅₀,” refers to that concentration of acompound at which a given activity, including binding of HBP polypeptideor other ligand and a functional activity of a receptor polypeptide, is50% of the maximum for that receptor activity measurable using the sameassay in the absence of compound. Stated differently, the “EC₅₀” is theconcentration of compound that gives 50% activation, when 100%activation is set at the amount of activity that does not increase withthe addition of more agonist. It should be noted that the “EC₅₀” of ananalog of HBP polypeptide will vary according to the identity of theanalogue used in the assay, for example, HBP polypeptide analogues canhave EC₅₀ values higher than, lower than or the same as HBP polypeptide.Therefore, where a HBP polypeptide analogue differs from HBPpolypeptide, one of skill in the art can determine the EC₅₀ for thatanalogue according to conventional methods. The EC₅₀ of a given HBPpolypeptide analogue is measured by performing an assay for the activityof a fixed amount of FPRL2 polypeptide polypeptide in the presence ofdoses of HBP polypeptide analogues that increase at least until theFPRL2 polypeptide response is saturated or maximal, and then plottingthe measured FPRL2 polypeptide activity versus the concentration of HBPpolypeptide analogues.

As used herein, the term “saturation” refers to the concentration of HBPpolypeptide or other ligand at which further increases in ligandconcentration fail to increase the binding of ligand or FPRL2polypeptide-specific signalling activity.

As used herein, the term “IC₅₀” is the concentration of an antagonist orinverse agonist that reduces the maximal activation of a FPRL2polypeptide receptor by 50%.

As used herein, the term “LD50” refers to the dose of a particular agentnecessary to kill 50% of the subjects to which it is administered.

As used herein, the term “decrease in binding” refers to a decrease ofat least 10% in the amount of ligand binding detected in a given assaywith a known or suspected modulator of FPRL2 polypeptide relative tobinding detected in an assay lacking that known or suspected modulator.

As used herein, the term “delivering,” when used in reference to a drugor agent, means the addition of the drug or agent to an assay mixture,or to a cell in culture. The term also refers to the administration ofthe drug or agent to an animal. Such administration can be, for example,by injection (in a suitable carrier, e.g., sterile saline or water) orby inhalation, or by an oral, transdermal, rectal, vaginal, or othercommon route of drug administration.

As used herein, the term “standard” refers to a sample taken from anindividual who is not affected by a disease or disorder characterized bydysregulation of FPRL2 polypeptide activity. The “standard” is used as areference for the comparison of FPRL2 mRNA or polypeptide levels andquality (i.e., mutant vs. wild type), as well as for the comparison ofFPRL2 polypeptide activities. A “standard” also encompasses a referencesequence, e.g., SEQ ID NO: 1 or SEQ ID NO: 2, with which sequences ofnucleic acids or their encoded polypeptides are compared.

As used herein, the term “amplifying,” when applied to a nucleic acidsequence, refers to a process whereby one or more copies of a nucleicacid sequence is generated from a template nucleic acid. A preferredmethod of “amplifying” is PCR or RT/PCR.

As used herein, the term “G-Protein coupled receptor,” or “GPCR” refersto a membrane-associated polypeptide with 7 alpha helical transmembranedomains. Functional GPCR's associate with a ligand or agonist and alsoassociate with and activate G-proteins. FPRL2 polypeptide is a GPCR.

As used herein, the term “antibody” is the conventional immunoglobulinmolecule, as well as fragments thereof which are also specificallyreactive with one of the subject polypeptides. Antibodies can befragmented using conventional techniques and the fragments screened forutility in the same manner as described herein below for wholeantibodies. For example, F(ab)₂ fragments can be generated by treatingantibody with pepsin. The resulting F(ab)₂ fragment can be treated toreduce disulfide bridges to produce Fab fragments. The antibody of thepresent invention is further intended to include bispecific,single-chain, and chimeric and humanised molecules having affinity for apolypeptide conferred by at least one CDR region of the antibody. Inpreferred embodiments, the antibody further comprises a label attachedthereto and able to be detected, (e.g., the label can be a radioisotope,fluorescent compound, chemiluminescent compound, enzyme, or enzymeco-factor). The antibodies, monoclonal or polyclonal and itshypervariable portion thereof (F(ab), F(ab′)2, etc.) as well as thehybridoma cell producing the antibodies are a further aspect of thepresent invention which find a specific industrial application in thefield of diagnostics and monitoring of specific diseases, preferably theones hereafter described.

Inhibitors and modulators according to the invention include but are notlimited to monoclonal or polyclonal antibodies or hypervariable portionsof the antibodies.

The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising portions of immunoglobulins of a differentorigin, wherein at least one portion is of human origin. Accordingly,the present invention relates to a humanized immunoglobulin which bindshuman FPRL2, said immunoglobulin comprising an antigen-binding region ofnonhuman origin (e.g., rodent) and at least a portion of animmunoglobulin of human origin (e.g., a human framework region, a humanconstant region or portion thereof). For example, the humanized antibodycan comprise portions derived from an immunoglobulin of nonhuman originwith the requisite specificity, such as a mouse, and from immunoglobulinsequences of human origin (e.g., a chimeric immunoglobulin), joinedtogether chemically by conventional techniques (e.g., synthetic) orprepared as contiguous polypeptide using genetic engineering techniques(e.g., DNA encoding the protein portions of the chimeric antibody can beexpresses to produce a contiguous polypeptide chain). Another example ofa humanized immunoglobulin of the present invention is an immunoglobulincontaining one or more immunoglobulin chains comprising a CDR ofnonhuman origin (e.g., one or more CDRs derived from an antibody ofnonhuman origin) and a framework region derived from a light and/orheavy chain of human origin (e.g., CDR-grafted antibodies with orwithout framework changes).

Such humanized immunoglobulins can be produced using synthetic and/orrecombinant nucleic acids to prepare genes (e.g., cDNA) encoding thedesired humanized chain. For example, nucleic acid (e.g., DNA) sequencescoding for humanized variable regions can be constructed using PCRmutagenesis methods to alter DNA sequences encoding a human or humanizedchain, such as a DNA template form a previously humanized variableregion (see e.g., Kamman, M., et al., Nucleic Acids Res., 17: 5404(1989); Sato, K., et al., Cancer Research, 53: 851-856 (1993);Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991);and Lewis, A. P. and J. S Crowe, Gene, 101: 297-302 (1991)). Using theseor other suitable methods, variants can also be readily produced. In oneembodiment, cloned variable regions can be mutagenized, and sequencesencoding variants with the desired specificity can be selected (e.g.,from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548;Hoogenboom et al., WO 93/06213, published Apr. 1, 1993; Knappik et al.,WO 97/08320, published Mar. 6, 1997)).

As used herein, the term “transgenic animal” refers to any animal,preferably a non-human mammal, bird, fish or an amphibian, in which oneor more of the cells of the animal contain heterologous nucleic acidintroduced by way of human intervention, such as by transgenictechniques well known in the art. The nucleic acid is introduced intothe cell, directly or indirectly by introduction into a precursor of thecell, by way of deliberate genetic manipulation, such as bymicroinjection or by infection with a recombinant virus. The termgenetic manipulation does not include classical cross-breeding, or invitro fertilization, but rather is directed to the introduction of arecombinant DNA molecule. This molecule may be integrated within achromosome, or it may be extra-chromosomally replicating DNA. In thetypical transgenic animals described herein, the transgene causes cellsto express a recombinant form of one of the subject polypeptide, e.g.either agonistic or antagonistic forms. However, transgenic animals inwhich the recombinant gene is silent are also contemplated, as forexample, the FLP or CRE recombinase dependent constructs describedbelow. Moreover, “transgenic animal” also includes those recombinantanimals in which gene disruption of one or more genes is caused by humanintervention, including both recombination and antisense techniques.

Sequences

The invention relates to the nucleotide (SEQ ID NO: 1) and amino acid(SEQ ID NO: 2) sequences encoding FPRL2 polypeptide (presented in FIG.1). The invention also relates to sequences that are homologous to thenucleotide and amino acid sequences encoding FPRL2 polypeptide.

Calculation of Sequence Homology

Sequence identity with respect to any of the sequences presented hereincan be determined by a simple “eyeball” comparison (i.e. a strictcomparison) of any one or more of the sequences with another sequence tosee if that other sequence has, for example, at least 80% sequenceidentity to the sequence(s).

Relative sequence identity can also be determined by commerciallyavailable computer programs that can calculate % identity between two ormore sequences using any suitable algorithm for determining identity,using for example default parameters. A typical example of such acomputer program is CLUSTAL. Other computer program methods to determineidentity and similarity between two sequences include but are notlimited to the GCG program package (Devereux et al 1984 Nucleic AcidsResearch 12: 387) and FASTA (Atschul et al 1990 J Molec Biol 403-410).

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example, when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software that can perform sequence comparisons include, but arenot limited to, the BLAST package (Ausubel et al., 1995, Short Protocolsin Molecular Biology, 3rd Edition, John Wiley & Sons), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (Ausubel et al., 1999 supra, pages 7-58 to 7-60).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix-the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied.It is preferred to use the public default values for the GCG package, orin the case of other software, the default matrix, such as BLOSUM62.

Advantageously, the BLAST algorithm is employed, with parameters set todefault values. The BLAST algorithm is described in detail athttp://www.ncbi.nih.gov/BLAST/blast_help.html, which is incorporatedherein by reference. The search parameters are defined as follows, andcan be advantageously set to the defined default parameters.

Advantageously, “substantial identity” when assessed by BLAST equates tosequences which match with an EXPECT value of at least about 7,preferably at least about 9 and most preferably 10 or more. The defaultthreshold for EXPECT in BLAST searching is usually 10.

BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, tblastn, andtblastx; these programs ascribe significance to their findings using thestatistical methods of Karlin and Altschul (Karlin and Altschul 1990,Proc. Matl. Acad. Sci. USA 87:2264-68; Karlin and Altschul, 1993, Proc.Natl. Acad. Sci. USA 90:5873-7; see http:www.ncbi.nihgov/BLAST/blast_help.html) with a few enhancements. The BLAST programsare tailored for sequence similarity searching, for example to identifyhomologues to a query sequence. For a discussion of basic issues insimilarity searching of sequence databases, see Altschul et al (1994)Nature Genetics 6:119-129.

The five BLAST programs available at http:www.ncbi.mm.nih.gov performthe following tasks: blastp—compares an amino acid query sequenceagainst a protein sequence database; blastn—compares a nucleotide querysequence against a nucleotide sequence database; blastx—compares thesix-frame conceptual translation products of a nucleotide query sequence(both strands) against a protein sequence database; tblastn—compares aprotein query sequence against a nucleotide sequence databasedynamically translated in all six reading frames (both strands);tblastx—compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

BLAST uses the following search parameters:

HISTOGRAM—Display a histogram of scores for each search; default is yes.(See parameter H in the BLAST Manual).

DESCRIPTIONS—Restricts the number of short descriptions of matchingsequences reported to the number specified; default limit is 100descriptions. (See parameter V in the manual page).

EXPECT—The statistical significance threshold for reporting matchesagainst database sequences; the default value is 10, such that 10matches are expected to be found merely by chance, according to thestochastic model of Karlin and Altschul (1990). If the statisticalsignificance ascribed to a match is greater than the EXPECT threshold,the match will not be reported. Lower EXPECT thresholds are morestringent, leading to fewer chance matches being reported. Fractionalvalues are acceptable. (See parameter E in the BLAST Manual).

CUTOFF—Cutoff score for reporting high-scoring segment pairs. Thedefault value is calculated from the EXPECT value (see above). HSPs arereported for a database sequence only if the statistical significanceascribed to them is at least as high as would be ascribed to a lone HSPhaving a score equal to the CUTOFF value. Higher CUTOFF values are morestringent, leading to fewer chance matches being reported. (Seeparameter S in the BLAST Manual). Typically, significance thresholds canbe more intuitively managed using EXPECT.

ALIGNMENTS—Restricts database sequences to the number specified forwhich high-scoring segment pairs (HSPs) are reported; the default limitis 50. If more database sequences than this happen to satisfy thestatistical significance threshold for reporting (see EXPECT and CUTOFFbelow), only the matches ascribed the greatest statistical significanceare reported. (See parameter B in the BLAST Manual).

MATRIX—Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTNand TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992).The valid alternative choices include: PAM40, PAM120, PAM250 andIDENTITY. No alternate scoring matrices are available for BLASTN;specifying the MATRIX directive in BLASTN requests returns an errorresponse.

STRAND—Restrict a TBLASTN search to just the top or bottom strand of thedatabase sequences; or restrict a BLASTN, BLASTX or TBLASTX search tojust reading frames on the top or bottom strand of the query sequence.

FILTER—Mask off segments of the query sequence that have lowcompositional complexity, as determined by the SEG program of Wootton &Federhen (1993) Computers and Chemistry 17:149-163, or segmentsconsisting of short-periodicity internal repeats, as determined by theXNU program of Clayerie & States (1993) Computers and Chemistry17:191-201, or, for BLASTN, by the DUST program of Tatusov and Lipman(see http:www.ncbi.nlm.nih.gov). Filtering can eliminate statisticallysignificant but biologically uninteresting reports from the blast output(e.g., hits against common acidic-, basic- or proline-rich regions),leaving the more biologically interesting regions of the query sequenceavailable for specific matching against database sequences.

Low complexity sequence found by a filter program is substituted usingthe letter “N” in nucleotide sequence (e.g., “NNNNNNNNNNNNN”) and theletter “X” in protein sequences (e.g., “XXXXXXXXX”).

Filtering is only applied to the query sequence (or its translationproducts), not to database sequences. Default filtering is DUST forBLASTN, SEG for other programs.

It is not unusual for nothing at all to be masked by SEG, XNU, or both,when applied to sequences in SWISS-PROT, so filtering should not beexpected to always yield an effect. Furthermore, in some cases,sequences are masked in their entirety, indicating that the statisticalsignificance of any matches reported against the unfiltered querysequence should be suspect.

NCBI-gi—Causes NCBI gi identifiers to be shown in the output, inaddition to the accession and/or locus name.

Most preferably, sequence comparisons are conducted using the simpleBLAST search algorithm provided at http:www.ncbi.nlm.nih.gov/BLAST. Insome embodiments of the present invention, no gap penalties are usedwhen determining sequence identity.

Cells

A cell that is useful according to the invention is preferably selectedfrom the group consisting of bacterial cells, yeast cells, insect cellsor mammalian cells.

A cell that is useful according to the invention can be any cell intowhich a nucleic acid sequence encoding a receptor according to theinvention can be introduced such that the receptor is expressed atnatural levels or above natural levels, as defined herein. Preferably areceptor of the invention that is expressed in a cell exhibits normal ornear normal pharmacology, as defined herein. Most preferably a receptorof the invention that is expressed in a cell comprises the nucleotiderepresented by SEQ ID NO: 1 or amino acid sequence represented by SEQ IDNO: 2 or a nucleotide or amino acid sequence that is at least 70%identical to the amino acid sequence represented by SEQ ID NO: 2.Preferably, a receptor of the invention that is expressed in a cell willbind HBP polypeptide.

According to a preferred embodiment of the present invention, a cell isselected from the group consisting of COS7-cells, a CHO cell, a LM (TK-)cell, a NIH-3T3 cell, HEK-293 cell, K-562 cell or a 1321N1 astrocytomacell but also other transfectable cell lines.

Assays

I. Assays for the Identification of Agents that Modulate the Activity ofFPRL2 Polypeptide

Agents that modulate the activity of FPRL2 polypeptide can be identifiedin a number of ways that take advantage of the newly discoveredinteraction of the receptor with HBP polypeptide. For example, theability to reconstitute FPRL2 polypeptide/HBP polypeptide binding eitherin vitro, on cultured cells or in vivo provides a target for theidentification of agents that disrupt that binding. Assays based ondisruption of binding can identify agents, such as small organicmolecules, from libraries or collections of such molecules.Alternatively, such assays can identify agents in samples or extractsfrom natural sources, e.g., plant, fungal or bacterial extracts or evenin human tissue samples (e.g., tumor tissue). In one aspect, theextracts can be made from cells expressing a library of variant nucleicacids, peptides or polypeptides. Modulators of FPRL2 polypeptide/HBPpolypeptide binding can then be screened using a binding assay or afunctional assay that measures downstream signalling through thereceptor.

Another approach that uses the FPRL2 polypeptide/HBP polypeptideinteraction more directly to identify agents that modulate FPRL2polypeptide function measures changes in FPRL2 polypeptide downstreamsignalling induced by candidate agents or candidate modulators. Thesefunctional assays can be performed in isolated cell membrane fractionsor on cells expressing the receptor on their surfaces.

The finding that HBP polypeptide is a ligand of the FPRL2 polypeptidereceptor permits screening assays to identify agonists, antagonists andinverse agonists of receptor activity. The screening assays have twogeneral approaches, detailed below. For the purposes of this section HBPpolypeptide (SEQ ID NO: 18) is used as an exemplary ligand. It should beunderstood, however, that any HBP polypeptide as defined herein can beused in the assays described.

1) Ligand binding assays, in which cells expressing FPRL2 polypeptide,membrane extracts from such cells, or immobilized lipid membranescomprising FPRL2 polypeptide are exposed to labelled and candidatecompound. Following incubation, the reaction mixture is measured forspecific binding of the labelled to the FPRL2 polypeptide receptor.Compounds that interfere with binding or displace labelled can beagonists, antagonists or inverse agonists of FPRL2 polypeptide activity.Subsequent functional analysis can then be performed on positivecompounds to determine in which of these categories they belong.

2) Functional assays, in which a signalling activity of FPRL2polypeptide is measured.

a) For agonist screening, cells expressing FPRL2 polypeptide ormembranes prepared from them are incubated with a candidate compound,and a signalling activity of FPRL2 polypeptide is measured. The activityinduced by compounds that modulate receptor activity is compared to thatinduced by the natural ligand, an HBP polypeptide. An agonist or partialagonist will have a maximal biological activity corresponding to atleast 10% of the maximal activity of HBP polypeptide when the agonist orpartial agonist is present at 1 mM or less, and preferably will have apotency which is at least as potent as HBP polypeptide.

b) For antagonist or inverse agonist screening, cells expressing FPRL2polypeptide or membranes isolated from them are assayed for signallingactivity in the presence of HBP polypeptide with or without a candidatecompound. Antagonists will reduce the level of HBPpolypeptide-stimulated receptor activity by at least 10%, relative toreactions lacking the antagonist in the presence of HBP polypeptide.Inverse agonists will reduce the constitutive activity of the receptorby at least 10%, relative to reactions lacking the inverse agonist.

c) For inverse agonist screening, cells expressing constitutive FPRL2polypeptide activity or membranes isolated from them are used in afunctional assay that measures an activity of the receptor in thepresence of a candidate compound. Inverse agonists are those compoundsthat reduce the constitutive activity of the receptor by at least 10%.Overexpression of FPRL2 polypeptide may lead to constitutive activation.FPRL2 polypeptide can be overexpressed by placing it under the controlof a strong constitutive promoter, e.g., the CMV early promoter.Alternatively, certain mutations of conserved GPCR amino acids or aminoacid domains tend to lead to constitutive activity. See for example:Kjelsberg et al., 1992, J. Biol. Chem. 267:1430; McWhinney et al., 2000.J. Biol. Chem. 275:2087; Ren et al., 1993, J. Biol. Chem. 268:16483;Samama et al., 1993, J. Biol. Chem. 268:4625; Parma et al., 1993, Nature365:649; Parma et al., 1998, J. Pharmacol. Exp. Ther. 286:85; and Parentet al., 1996, J. Biol. Chem. 271:7949.

Ligand Binding and Displacement Assays:

As noted in (1) above, one can use FPRL2 polypeptides expressed on acell, or isolated membranes containing receptor polypeptides, along withHBP polypeptide in order to screen for compounds that inhibit thebinding of HBP polypeptide to FPRL2 polypeptide. For the purposes ofthis section, HBP polypeptide (SEQ ID NO: 18) is used as an exemplaryligand. It should be understood however that any HBP polypeptide asdefined herein can be used in the assays described.

For displacement experiments, cells expressing a FPRL2 polypeptide(generally 25,000 cells per assay or 1 to 100 μg of membrane extracts)are incubated in binding buffer with labelled HBP polypeptide in thepresence or absence of increasing concentrations of a candidatemodulator. To validate and calibrate the assay, control competitionreactions using increasing concentrations of unlabeled HBP polypeptidecan be performed. After incubation, cells are washed extensively, andbound, labelled HBP polypeptide is measured as appropriate for the givenlabel (e.g., scintillation counting, fluorescence, etc.). A decrease ofat least 10% in the amount of labelled HBP polypeptide bound in thepresence of candidate modulator indicates displacement of binding by thecandidate modulator. Candidate modulators are considered to bindspecifically in this or other assays described herein if they displace50% of labelled HBP polypeptide (sub-saturating HBP polypeptide dose) ata concentration of 1 mM or less.

Alternatively, binding or displacement of binding can be monitored bysurface plasmon resonance (SPR). Surface plasmon resonance assays can beused as a quantitative method to measure binding between two moleculesby the change in mass near an immobilized sensor caused by the bindingor loss of binding of HBP polypeptide from the aqueous phase to a FPRL2polypeptide immobilized in a membrane on the sensor. This change in massis measured as resonance units versus time after injection or removal ofthe HBP polypeptide or candidate modulator and is measured using aBiacore Biosensor (Biacore AB). FPRL2 polypeptide can be immobilized ona sensor chip (for example, research grade CM5 chip; Biacore AB) in athin film lipid membrane according to methods described by Salamon etal. (Salamon et al., 1996, Biophys J. 71: 283-294; Salamon et al., 2001,Biophys. J. 80: 1557-1567; Salamon et al., 1999, Trends Biochem. Sci.24: 213-219, each of which is incorporated herein by reference.). Sarrioet al. demonstrated that SPR can be used to detect ligand binding to theGPCR A(1) adenosine receptor immobilized in a lipid layer on the chip(Sarrio et al., 2000, Mol. Cell. Biol. 20: 5164-5174, incorporatedherein by reference). Conditions for HBP polypeptide binding to FPRL2polypeptide in an SPR assay can be fine-tuned by one of skill in the artusing the conditions reported by Sarrio et al. as a starting point.

SPR can assay for modulators of binding in at least two ways. First, HBPpolypeptide can be pre-bound to immobilized FPRL2 polypeptidepolypeptide, followed by injection of candidate modulator at aconcentration ranging from 0.1 nM to 1 μM. Displacement of the bound HPBpolypeptide can be quantitated, permitting detection of modulatorbinding. Alternatively, the membrane-bound FPRL2 polypeptide can bepre-incubated with candidate modulator and challenged with HBPpolypeptide. A difference in HBP polypeptide binding to the FPRL2polypeptide exposed to modulator relative to that on a chip notpre-exposed to modulator will demonstrate binding or displacement of HBPpolypeptide in the presence of modulator. In either assay, a decrease of10% or more in the amount of HBP polypeptide bound in the presence ofcandidate modulator, relative to the amount of a HBP polypeptide boundin the absence of candidate modulator indicates that the candidatemodulator inhibits the interaction of FPRL2 polypeptide and HBPpolypeptide.

Another method of detecting inhibition of binding of HBP polypeptide toFPRL2 polypeptide uses fluorescence resonance energy transfer (FRET).FRET is a quantum mechanical phenomenon that occurs between afluorescence donor (D) and a fluorescence acceptor (A) in closeproximity to each other (usually <100 A of separation) if the emissionspectrum of D overlaps with the excitation spectrum of A. The moleculesto be tested, e.g. HBP polypeptide and a FPRL2 polypeptide, are labelledwith a complementary pair of donor and acceptor fluorophores. Whilebound closely together by the FPRL2 polypeptide: HBP polypeptideinteraction, the fluorescence emitted upon excitation of the donorfluorophore will have a different wavelength than that emitted inresponse to that excitation wavelength when the HBP polypeptide andFPRL2 polypeptide are not bound, providing for quantitation of boundversus unbound molecules by measurement of emission intensity at eachwavelength. Donor fluorophores with which to label the FPRL2 polypeptideare well known in the art. Of particular interest are variants of the A.victoria GFP known as Cyan FP (CFP, Donor (I)) and Yellow FP (YFP,Acceptor (A)). As an example, the YFP variant can be made as a fusionprotein with FPRL2 polypeptide. Vectors for the expression of GFPvariants as fusions (Clontech) as well as fluorophore-labeled HBPpolypeptide compounds (Molecular Probes) are known in the art. Theaddition of a candidate modulator to the mixture of labelled HBPpolypeptide and YFP-FPRL2 protein will result in an inhibition of energytransfer evidenced by, for example, a decrease in YFP fluorescencerelative to a sample without the candidate modulator. In an assay usingFRET for the detection of FPRL2 polypeptide: HBP polypeptideinteraction, a 10% or greater decrease in the intensity of fluorescentemission at the acceptor wavelength in samples containing a candidatemodulator, relative to samples without the candidate modulator,indicates that the candidate modulator inhibits the FPRL2 polypeptide:HBP polypeptide interaction.

A variation on FRET uses fluorescence quenching to monitor molecularinteractions. One molecule in the interacting pair can be labelled witha fluorophore, and the other with a molecule that quenches thefluorescence of the fluorophore when brought into close apposition withit. A change in fluorescence upon excitation is indicative of a changein the association of the molecules tagged with the fluorophore:quencherpair. Generally, an increase in fluorescence of the labelled FPRL2polypeptide is indicative that the HBP polypeptide molecule bearing thequencher has been displaced. For quenching assays, a 10% or greaterincrease in the intensity of fluorescent emission in samples containinga candidate modulator, relative to samples without the candidatemodulator, indicates that the candidate modulator inhibits FPRL2polypeptide: HBP polypeptide interaction.

In addition to the surface plasmon resonance and FRET methods,fluorescence polarization measurement is useful to quantitate binding.The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Complexes,such as those formed by FPRL2 polypeptide associating with afluorescently labelled HBP polypeptide, have higher polarization valuesthan uncomplexed, labelled HBP polypeptide. The inclusion of a candidateinhibitor of the FPRL2 polypeptide: HBP polypeptide interaction resultsin a decrease in fluorescence polarization, relative to a mixturewithout the candidate inhibitor, if the candidate inhibitor disrupts orinhibits the interaction of FPRL2 polypeptide with HBP polypeptide.Fluorescence polarization is well suited for the identification of smallmolecules that disrupt the formation of receptor:ligand complexes. Adecrease of 10% or more in fluorescence polarization in samplescontaining a candidate modulator, relative to fluorescence polarizationin a sample lacking the candidate modulator, indicates that thecandidate modulator inhibits FPRL2 polypeptide: HBP polypeptideinteraction.

Another alternative for monitoring FPRL2 polypeptide: HBP polypeptideinteractions uses a biosensor assay. ICS biosensors have been describedin the art (Australian Membrane Biotechnology Research Institute;www.ambri.com.au/; Cornell B, Braach-Maksvytis V, King L, Osman P,Raguse B, Wieczorek L, and Pace R. “A biosensor that uses ion-channelswitches” Nature 1997, 387, 580). In this technology, the association ofFPRL2 polypeptide and its ligand is coupled to the closing ofgramacidin-facilitated ion channels in suspended membrane bilayers andthus to a measurable change in the admittance (similar to impedence) ofthe biosensor. This approach is linear over six orders of magnitude ofadmittance change and is ideally suited for large scale, high throughputscreening of small molecule combinatorial libraries. A 10% or greaterchange (increase or decrease) in admittance in a sample containing acandidate modulator, relative to the admittance of a sample lacking thecandidate modulator, indicates that the candidate modulator inhibits theinteraction of FPRL2 polypeptide and HBP polypeptide. It is important tonote that in assays testing the interaction of FPRL2 polypeptide withHBP polypeptide, it is possible that a modulator of the interaction neednot necessarily interact directly with the domain(s) of the proteinsthat physically interact with HBP polypeptide. It is also possible thata modulator will interact at a location removed from the site ofinteraction and cause, for example, a conformational change in the FPRL2polypeptide. Modulators (inhibitors or agonists) that act in this mannerare nonetheless of interest as agents to modulate the activity of FPRL2polypeptide.

It should be understood that any of the binding assays described hereincan be performed with a non-HBP polypeptide ligand (for example,agonist, antagonist, etc.) of FPRL2 polypeptide, e.g., a small moleculeidentified as described herein or HBP polypeptide analogues includingbut not limited to any of the HBP polypeptide analogues, a natural orsynthetic peptide, a polypeptide, an antibody or antigen-bindingfragment thereof, a lipid, a carbohydrate, and a small organic molecule.

Any of the binding assays described can be used to determine thepresence of an agent in a sample, e.g., a tissue sample, that binds tothe FPRL2 polypeptide receptor molecule, or that affects the binding ofHBP polypeptide to the receptor. To do so, FPRL2 polypeptide is reactedwith HBP polypeptide or another ligand in the presence or absence of thesample, and HBP polypeptide or ligand binding is measured as appropriatefor the binding assay being used. A decrease of 10% or more in thebinding of HBP polypeptide or other ligand indicates that the samplecontains an agent that modulates HBP polypeptide or ligand binding tothe receptor polypeptide.

Functional Assays of Receptor Activity

i. GTPase/GTP Binding Assays:

For GPCRs such as FPRL2 polypeptide, a measure of receptor activity isthe binding of GTP by cell membranes containing receptors. In the methoddescribed by Traynor and Nahorski, 1995, Mol. Pharmacol. 47: 848-854,incorporated herein by reference, one essentially measures G-proteincoupling to membranes by detecting the binding of labelled GTP. For GTPbinding assays, membranes isolated from cells expressing the receptorare incubated in a buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl,and 10 mM MgCl2, 80 pM ³⁵S-GTPγS and 3 μM GDP. The assay mixture isincubated for 60 minutes at 30° C., after which unbound labelled GTP isremoved by filtration onto GF/B filters. Bound, labelled GTP is measuredby liquid or solid (SPA, see below) scintillation counting. In order toassay for modulation of HBP polypeptide-induced FPRL2 polypeptideactivity, membranes prepared from cells expressing a FPRL2 polypeptideare mixed with HBP polypeptide, and the GTP binding assay is performedin the presence and absence of a candidate modulator of FPRL2polypeptide activity. A decrease of 10% or more in labelled GTP bindingas measured by scintillation counting in an assay of this kindcontaining a candidate modulator, relative to an assay without themodulator, indicates that the candidate modulator inhibits FPRL2polypeptide activity. A similar GTP-binding assay can be performedwithout HBP polypeptide to identify compounds that act as agonists. Inthis case, HBP polypeptide-stimulated GTP binding is used as a standard.A compound is considered an agonist if it induces at least 50, 40, 30,or preferably 20% of the level of GTP binding induced by HBP polypeptidewhen the compound is present at 1 μM or less, and preferably will inducea level the same as or higher than that induced by HBP polypeptide.

Scintillation Proximity Assay (SPA) is an homogeneous screeningtechnology applied to receptor binding assays by immobilizing receptorsdirectly onto SPA beads via a suitable coupling method. Onceimmobilized, the receptor is close enough to the bead so that, should asuitably radiolabelled ligand bind to the receptor, it will be in closeenough proximity to stimulate the bead to emit light. Any unboundradioligand is too distant from the bead to transfer energy and goesundetected. The bead, therefore, only detects the population of ligandmolecules which are receptor bound. The discrimination of binding byproximity means that no separation of bound and free ligand is required,as in traditional methods. The method is generally applicable to [³H],[¹²⁵I], [³⁵S] ligands. Approaches involving antibodies and biotinylationcan be used for soluble receptors

GTPase activity is measured by incubating the membranes containing aFPRL2 polypeptide with γ³²P-GTP. Active GTPase will release the label asinorganic phosphate, which is detected by separation of free inorganicphosphate in a 5% suspension of activated charcoal in 20 mM H₃PO₄,followed by scintillation counting. Controls include assays usingmembranes isolated from cells not expressing FPRL2 polypeptide(mock-transfected), in order to exclude possible non-specific effects ofthe candidate compound.

In order to assay for the effect of a candidate modulator on FPRL2polypeptide-regulated GTPase activity, membrane samples are incubatedwith HBP polypeptide, with or without the modulator, followed by theGTPase assay. A change (increase or decrease) of 10% or more in thelevel of GTP binding or GTPase activity relative to samples withoutmodulator is indicative of FPRL2 polypeptide modulation by a candidatemodulator.

ii. Downstream Pathway Activation Assays:

a. Calcium Flux—The Aeguorin-Based Assay.

The aequorin assay takes advantage of the responsiveness ofmitochondrial apoaequorin to intracellular calcium release induced bythe activation of GPCRs (Stables et al., 1997, Anal. Biochem.252:115-126; Detheux et al., 2000, J. Exp. Med., 192 1501-1508; both ofwhich are incorporated herein by reference). Briefly, FPRL2polypeptide-expressing clones are transfected to coexpress mitochondrialapoaequorin and Gα16. Cells are incubated with 5 μM Coelenterazine H(Molecular Probes) for 4 hours at room temperature, washed in DMEM-F12culture medium and resuspended at a concentration of 0.5×10⁶ cells/ml.Cells are then mixed with test agonist molecules and light emission bythe aequorin is recorded with a luminometer for 30 sec. Results areexpressed as Relative Light Units (RLU). Controls include assays usingmembranes isolated from cells not expressing FPRL2 polypeptide (mocktransfected), in order to exclude possible non-specific effects of thecandidate compound.

Aequorin activity or intracellular calcium levels are “changed” if lightintensity increases or decreases by 10% or more in a sample of cells,expressing a FPRL2 polypeptide and treated with a candidate modulator,relative to a sample of cells expressing the FPRL2 polypeptide but nottreated with the candidate modulator or relative to a sample of cellsnot expressing the FPRL2 polypeptide (mock-transfected cells) buttreated with the candidate modulator.

When performed in the absence of HBP polypeptide, the assay can be usedto identify an agonist of FPRL2 polypeptide activity. When the assay isperformed in the presence of HBP polypeptide, it can be used to assayfor an antagonist.

b. Adenylate Cyclase Assay:

Assays for adenylate cyclase activity are described by Kenimer &Nirenberg, 1981, Mol. Pharmacol. 20: 585-591, incorporated herein byreference. That assay is a modification of the assay taught by Solomonet al., 1974, Anal. Biochem. 58: 541-548, also incorporated herein byreference. Briefly, 1001 reactions contain 50 mM Tris-Hcl (pH 7.5), 5 mMMgCl₂, 20 mM creatine phosphate (disodium salt), 10 units (71 μg ofprotein) of creatine phosphokinase, 1 mM α-³²P-ATP (tetrasodium salt, 2μCi), 0.5 mM cyclic AMP, G-³H-labeled cyclic AMP (approximately 10,000cpm), 0.5 mM Ro20-1724, 0.25% ethanol, and 50-200 μg of proteinhomogenate to be tested (i.e., homogenate from cells expressing or notexpressing a FPRL2 polypeptide, treated or not treated with HBPpolypeptide with or without a candidate modulator). Reaction mixturesare generally incubated at 37° C. for 60 minutes. Following incubation,reaction mixtures are deproteinized by the addition of 0.9 ml of cold 6%trichloroacetic acid. Tubes are centrifuged at 1800×g for 20 minutes andeach supernatant solution is added to a Dowex AG50W-X4 column. The cAMPfraction from the column is eluted with 4 ml of 0.1 mM imidazole-HCl (pH7.5) into a counting vial. Assays should be performed in triplicate.Control reactions should also be performed using protein homogenate fromcells that do not express a FPRL2 polypeptide.

According to the invention, adenylate cyclase activity is “changed” ifit increases or decreases by 10% or more in a sample taken from cellstreated with a candidate modulator of FPRL2 polypeptide activity,relative to a similar sample of cells not treated with the candidatemodulator or relative to a sample of cells not expressing the FPRL2polypeptide (mock-transfected cells) but treated with the candidatemodulator.

c. cAMP Assay:

Intracellular or extracellular cAMP is measured using a cAMPradioimmunoassay (RIA) or cAMP binding protein according to methodswidely known in the art. For example, Horton & Baxendale, 1995, MethodsMol. Biol. 41: 91-105, which is incorporated herein by reference,describes an RIA for cAMP.

A number of kits for the measurement of cAMP are commercially available,such as the High Efficiency Fluorescence Polarization-based homogeneousassay marketed by LJL Biosystems and NEN Life Science Products. Controlreactions should be performed using extracts of mock-transfected cellsto exclude possible non-specific effects of some candidate modulators.

The level of cAMP is “changed” if the level of cAMP detected in cells,expressing a FPRL2 polypeptide and treated with a candidate modulator ofFPRL2 polypeptide activity (or in extracts of such cells), using theRIA-based assay of Horton & Baxendale, 1995, supra, increases ordecreases by at least 10% relative to the cAMP level in similar cellsnot treated with the candidate modulator.

d. Phospholipid Breakdown, DAG Production and Inositol TrisphosphateLevels:

Receptors that activate the breakdown of phospholipids can be monitoredfor changes due to the activity of known or suspected modulators ofFPRL2 polypeptide by monitoring phospholipid breakdown, and theresulting production of second messengers DAG and/or inositoltrisphosphate (IP₃). Methods of detecting each of these are described inPhospholipid Signalling Protocols, edited by Ian M. Bird. Totowa, N.J.,Humana Press, 1998, which is incorporated herein by reference. See alsoRudolph et al., 1999, J. Biol. Chem. 274: 11824-11831, incorporatedherein by reference, which also describes an assay forphosphatidylinositol breakdown. Assays should be performed using cellsor extracts of cells expressing FPRL2 polypeptide, treated or nottreated with HBP polypeptide with or without a candidate modulator.Control reactions should be performed using mock-transfected cells, orextracts from them in order to exclude possible non-specific effects ofsome candidate modulators.

According to the invention, phosphatidylinositol breakdown, anddiacylglycerol and/or inositol trisphosphate levels are “changed” ifthey increase or decrease by at least 10% in a sample from cellsexpressing a FPRL2 polypeptide and treated with a candidate modulator,relative to the level observed in a sample from cells expressing a FPRL2polypeptide that is not treated with the candidate modulator.

e. PKC Activation Assays:

Growth factor receptor tyrosine kinases can signal via a pathwayinvolving activation of Protein Kinase C (PKC), which is a family ofphospholipid- and calcium-activated protein kinases. PKC activationultimately results in the transcription of an array of proto-oncogenetranscription factor-encoding genes, including c-fos, c-myc and c-jun,proteases, protease inhibitors, including collagenase type I andplasminogen activator inhibitor, and adhesion molecules, includingintracellular adhesion molecule I (ICAM I). Assays designed to detectincreases in gene products induced by PKC can be used to monitor PKCactivation and thereby receptor activity. In addition, the activity ofreceptors that signal via PKC can be monitored through the use ofreporter gene constructs driven by the control sequences of genesactivated by PKC activation. This type of reporter gene-based assay isdiscussed in more detail below.

For a more direct measure of PKC activity, the method of Kikkawa et al.,1982, J. Biol. Chem. 257: 13341, incorporated herein by reference, canbe used. This assay measures phosphorylation of a PKC substrate peptide,which is subsequently separated by binding to phosphocellulose paper.This PKC assay system can be used to measure activity of purifiedkinase, or the activity in crude cellular extracts. Protein kinase Csample can be diluted in 20 mM HEPES/2 mM DTT immediately prior toassay.

The substrate for the assay is the peptide Ac-FKKSFKL-NH2 (SEQ ID NO:11), derived from the myristoylated alanine-rich protein kinase Csubstrate protein (MARCKS). The K_(m) of the enzyme for this peptide isapproximately 50 μM. Other basic, protein kinase C-selective peptidesknown in the art can also be used, at a concentration of at least 2-3times their K_(m). Cofactors required for the assay include calcium,magnesium, ATP, phosphatidylserine and diacylglycerol. Depending uponthe intent of the user, the assay can be performed to determine theamount of PKC present (activating conditions) or the amount of activePKC present (non-activating conditions). For most purposes according tothe invention, non-activating conditions will be used, such that thePKC, that is active in the sample when it is isolated, is measured,rather than measuring the PKC that can be activated. For non-activatingconditions, calcium is omitted from the assay in favor of EGTA.

The assay is performed in a mixture containing 20 mM HEPES, pH 7.4, 1-2mM DTT, 5 mM MgCl₂, 100 μM ATP, ˜1 μCi γ-³²P-ATP, 100 μg/ml peptidesubstrate (˜100 μM), 140 μM/3.8 μM phosphatidylserine/diacylglycerolmembranes, and 100 μM calcium (or 500 μM EGTA). 48 μl of sample, dilutedin 20 mM HEPES, pH 7.4, 2 mM DTT is used in a final reaction volume of80 μl. Reactions are performed at 30° C. for 5-10 minutes, followed byaddition of 25 μl of 100 mM ATP, 100 mM EDTA, pH 8.0, which stops thereactions.

After the reaction is stopped, a portion (85 μl) of each reaction isspotted onto a Whatman P81 cellulose phosphate filter, followed bywashes: four times 500 ml in 0.4% phosphoric acid, (5-10 min per wash);and a final wash in 500 ml 95% EtOH, for 2-5 min. Bound radioactivity ismeasured by scintillation counting. Specific activity (cpm/nmol) of thelabelled ATP is determined by spotting a sample of the reaction onto P81paper and counting without washing. Units of PKC activity, defined asnmol phosphate transferred per min, are calculated as follows:

${{The}\mspace{14mu}{activity}},{{{in}\mspace{14mu}{UNITS}\mspace{14mu}\left( {{nmol}\text{/}\min} \right)\mspace{14mu}{is}}:={\frac{\left( {{cpm}\mspace{14mu}{on}\mspace{14mu}{paper}} \right) \times \left( \frac{105\mspace{14mu}\mu\; l\mspace{14mu}{total}}{85\mspace{14mu}\mu\; l\mspace{14mu}{spotted}} \right)}{\left( {{{assay}\mspace{14mu}{time}},\min} \right)\left( {{specific}\mspace{14mu}{activity}\mspace{14mu}{of}\mspace{14mu}{ATP}\mspace{14mu}{cpm}\text{/}{nmol}} \right)}.}}$

An alternative assay can be performed using a Protein Kinase C Assay Kitsold by PanVera (Cat. # P2747).

Assays are performed on extracts from cells expressing a FPRL2polypeptide, treated or not treated with HBP polypeptide with or withouta candidate modulator. Control reactions should be performed usingmock-transfected cells, or extracts from them in order to excludepossible non-specific effects of some candidate modulators.

According to the invention, PKC activity is “changed” by a candidatemodulator when the units of PKC measured by either assay described aboveincrease or decrease by at least 10%, in extracts from cells expressingFPRL2 polypeptide and treated with a candidate modulator, relative to areaction performed on a similar sample from cells not treated with acandidate modulator.

f. Kinase Assays:

MAP kinase activity can be assayed using any of several kits availablecommercially, for example, the p38 MAP Kinase assay kit sold by NewEngland Biolabs (Cat # 9820) or the FlashPlate™ MAP Kinase assays soldby Perkin-Elmer Life Sciences.

MAP Kinase activity is “changed” if the level of activity is increasedor decreased by 10% or more in a sample from cells, expressing a FPRL2polypeptide, treated with a candidate modulator relative to MAP kinaseactivity in a sample from similar cells not treated with the candidatemodulator.

Direct assays for tyrosine kinase activity using known synthetic ornatural tyrosine kinase substrates and labelled phosphate are wellknown, as are similar assays for other types of kinases (e.g., Ser/Thrkinases). Kinase assays can be performed with both purified kinases andcrude extracts prepared from cells expressing a FPRL2 polypeptide,treated with or without HBP polypeptide, with or without a candidatemodulator. Control reactions should be performed using mock-transfectedcells, or extracts from them in order to exclude possible non-specificeffects of some candidate modulators. Substrates can be eitherfull-length protein or synthetic peptides representing the substrate.Pinna & Ruzzene (1996, Biochem. Biophys. Acta 1314: 191-225,incorporated herein by reference) list a number of phosphorylationsubstrate sites useful for detecting kinase activities. A number ofkinase substrate peptides are commercially available. One that isparticularly useful is the “Src-related peptide,” RRLIEDAEYAARG (SEQ IDNO: 12; available from Sigma #A7433), which is a substrate for manyreceptor and nonreceptor tyrosine kinases. Because the assay describedbelow requires binding of peptide substrates to filters, the peptidesubstrates should have a net positive charge to facilitate binding.Generally, peptide substrates should have at least 2 basic residues anda free amino terminus. Reactions generally use a peptide concentrationof 0.7-1.5 mM.

Assays are generally carried out in a 25 μl volume comprising 5 μl of 5×kinase buffer (5 mg/mL BSA, 150 mM Tris-Cl (pH 7.5), 100 mM MgCl₂;depending upon the exact kinase assayed for, MnCl₂ can be used in placeof or in addition to the MgCl₂), 5 μl of 1.0 mM ATP (0.2 mM finalconcentration), γ-32P-ATP (100-500 cpm/pmol), 3 μl of 10 mM peptidesubstrate (1.2 mM final concentration), cell extract containing kinaseto be tested (cell extracts used for kinase assays should contain aphosphatase inhibitor (e.g. 0.1-1 mM sodium orthovanadate)), and H₂O to25 μl. Reactions are performed at 30° C., and are initiated by theaddition of the cell extract.

Kinase reactions are performed for 30 seconds to about 30 minutes,followed by the addition of 45 μl of ice-cold 10% trichloroacetic acid(TCA). Samples are spun for 2 minutes in a microcentrifuge, and 35 μl ofthe supernatant is spotted onto Whatman P81 cellulose phosphate filtercircles. The filters are washed three times with 500 ml cold 0.5%phosphoric acid, followed by one wash with 200 ml of acetone at roomtemperature for 5 minutes. Filters are dried and incorporated ³²P ismeasured by scintillation counting. The specific activity of ATP in thekinase reaction (e.g., in cpm/pmol) is determined by spotting a smallsample (2-5 μl) of the reaction onto a P81 filter circle and countingdirectly, without washing. Counts per minute obtained in the kinasereaction (minus blank) are then divided by the specific activity todetermine the moles of phosphate transferred in the reaction.

Tyrosine kinase activity is “changed” if the level of kinase activity isincreased or decreased by 10% or more in a sample from cells, expressinga FPRL2 polypeptide, treated with a candidate modulator relative tokinase activity in a sample from similar cells not treated with thecandidate modulator.

g. Transcriptional Reporters for Downstream Pathway Activation:

The intracellular signal initiated by binding of an agonist to areceptor, e.g., FPRL2 polypeptide, sets in motion a cascade ofintracellular events, the ultimate consequence of which is a rapid anddetectable change in the transcription or translation of one or moregenes. The activity of the receptor can therefore be monitored bydetecting the expression of a reporter gene driven by control sequencesresponsive to FPRL2 activation.

As used herein “promoter” refers to the transcriptional control elementsnecessary for receptor-mediated regulation of gene expression, includingnot only the basal promoter, but also any enhancers ortranscription-factor binding sites necessary for receptor-regulatedexpression. By selecting promoters that are responsive to theintracellular signals resulting from agonist binding, and operativelylinking the selected promoters to reporter genes whose transcription,translation or ultimate activity is readily detectable and measurable,the transcription based reporter assay provides a rapid indication ofwhether a given receptor is activated.

Reporter genes such as luciferase, CAT, GFP, β-lactamase orβ-galactosidase are well known in the art, as are assays for thedetection of their products.

Genes particularly well suited for monitoring receptor activity are the“immediate early” genes, which are rapidly induced, generally withinminutes of contact between the receptor and the effector protein orligand. The induction of immediate early gene transcription does notrequire the synthesis of new regulatory proteins. In addition to rapidresponsiveness to ligand binding, characteristics of preferred genesuseful for making reporter constructs include: low or undetectableexpression in quiescent cells; induction that is transient andindependent of new protein synthesis; subsequent shut-off oftranscription requires new protein synthesis; and mRNAs transcribed fromthese genes have a short half-life. It is preferred, but not necessarythat a transcriptional control element have all of these properties forit to be useful.

An example of a gene that is responsive to a number of different stimuliis the c-fos proto-oncogene. The c-fos gene is activated in aprotein-synthesis-independent manner by growth factors, hormones,differentiation-specific agents, stress, and other known inducers ofcell surface proteins. The induction of c-fos expression is extremelyrapid, often occurring within minutes of receptor stimulation. Thischaracteristic makes the c-fos regulatory regions particularlyattractive for use as a reporter of receptor activation.

The c-fos regulatory elements include (see, Verma et al., 1987, Cell 51:513-514): a TATA box that is required for transcription initiation; twoupstream elements for basal transcription, and an enhancer, whichincludes an element with dyad symmetry and which is required forinduction by TPA, serum, EGF, and PMA.

The 20 bp c-fos transcriptional enhancer element located between −317and −298 bp upstream from the c-fos mRNA cap site, is essential forserum induction in serum starved NIH 3T3 cells. One of the two upstreamelements is located at −63 to −57 and it resembles the consensussequence for cAMP regulation.

The transcription factor CREB (cyclic AMP responsive element bindingprotein) is, as the name implies, responsive to levels of intracellularcAMP. Therefore, the activation of a receptor that signals viamodulation of cAMP levels can be monitored by detecting either thebinding of the transcription factor, or the expression of a reportergene linked to a CREB-binding element (termed the CRE, or cAMP responseelement). The DNA sequence of the CRE is TGACGTCA. Reporter constructsresponsive to CREB binding activity are described in U.S. Pat. No.5,919,649.

Other promoters and transcriptional control elements, in addition to thec-fos elements and CREB-responsive constructs, include the vasoactiveintestinal peptide (VIP) gene promoter (cAMP responsive; Fink et al.,1988, Proc. Natl. Acad. Sci. 85:6662-6666); the somatostatin genepromoter (cAMP responsive; Montminy et al., 1986, Proc. Natl. Acad. Sci.8.3:6682-6686); the proenkephalin promoter (responsive to cAMP,nicotinic agonists, and phorbol esters; Comb et al., 1986, Nature323:353-356); the phosphoenolpyruvate carboxy-kinase (PEPCK) genepromoter (cAMP responsive; Short et al., 1986, J. Biol. Chem.261:9721-9726).

Additional examples of transcriptional control elements that areresponsive to changes in GPCR activity include, but are not limited tothose responsive to the AP-1 transcription factor and those responsiveto NF-κB activity. The consensus AP-1 binding site is the palindromeTGA(C/G)TCA (Lee et al., 1987, Nature 325: 368-372; Lee et al., 1987,Cell 49: 741-752). The AP-1 site is also responsible for mediatinginduction by tumor promoters such as the phorbol ester12-O-tetradecanoylphorbol-beta-acetate (TPA), and are thereforesometimes also referred to as a TRE, for TPA-response element. AP-1activates numerous genes that are involved in the early response ofcells to growth stimuli. Examples of AP-1-responsive genes include, butare not limited to the genes for Fos and Jun (which proteins themselvesmake up AP-1 activity), Fos-related antigens (Fra) 1 and 2, IκBα,ornithine decarboxylase, and annexins I and II.

The NF-κB binding element has the consensus sequence GGGGACTTTCC (SEQ IDNO: 13). A large number of genes have been identified as NF-κBresponsive, and their control elements can be linked to a reporter geneto monitor GPCR activity. A small sample of the genes responsive toNF-κB includes those encoding IL-1β (Hiscott et al., 1993, Mol. Cell.Biol. 13: 6231-6240), TNF-α (Shakhov et al., 1990, J. Exp. Med. 171:35-47), CCR5 (Liu et al., 1998, AIDS Res. Hum. Retroviruses 14:1509-1519), P-selection (Pan & McEver, 1995, J. Biol. Chem. 270:23077-23083), Fas ligand (Matsui et al., 1998, J. Immunol. 161:3469-3473), GM-CSF (Schreck & Baeuerle, 1990, Mol. Cell. Biol. 10:1281-1286) and IκBα (Haskill et al., 1991, Cell 65: 1281-1289). Each ofthese references is incorporated herein by reference. Vectors encodingNF-κB-responsive reporters are also known in the art or can be readilymade by one of skill in the art using, for example, synthetic NF-κBelements and a minimal promoter, or using the NF-κB responsive sequencesof a gene known to be subject to NF-κB regulation. Further, NF-κBresponsive reporter constructs are commercially available from, forexample, CLONTECH.

A given promoter construct should be tested by exposing FPRL2polypeptide-expressing cells, transfected with the construct, to HBPpolypeptide. An increase of at least two-fold in the expression ofreporter in response to HBP polypeptide indicates that the reporter isan indicator of FPRL2 polypeptide activity.

In order to assay FPRL2 polypeptide activity with a transcriptionalreporter construct, cells that stably express a FPRL2 polypeptide arestably transfected with the reporter construct. To screen for agonists,the cells are left untreated, exposed to candidate modulators, orexposed to HBP polypeptide, and expression of the reporter is measured.The HBP polypeptide-treated cultures serve as a standard for the levelof transcription induced by a known agonist. An increase of at least 50%in reporter expression in the presence of a candidate modulatorindicates that the candidate is a modulator of FPRL2 polypeptideactivity. An agonist will induce at least as much, and preferably thesame amount or greater reporter expression than HBP polypeptide alone.This approach can also be used to screen for inverse agonists wherecells express a FPRL2 polypeptide at levels such that there is anelevated basal activity of the reporter in the absence of HBPpolypeptide or another agonist. A decrease in reporter activity of 10%or more in the presence of a candidate modulator, relative to itsabsence, indicates that the compound is an inverse agonist.

To screen for antagonists, the cells expressing FPRL2 polypeptide andcarrying the reporter construct are exposed to HBP polypeptide (oranother agonist) in the presence and absence of candidate modulator. Adecrease of 10% or more in reporter expression in the presence ofcandidate modulator, relative to the absence of the candidate modulator,indicates that the candidate is a modulator of FPRL2 polypeptideactivity.

Controls for transcription assays include cells not expressing FPRL2polypeptide but carrying the reporter construct, as well as cells with apromoterless reporter construct. Compounds that are identified asmodulators of FPRL2 polypeptide-regulated transcription should also beanalyzed to determine whether they affect transcription driven by otherregulatory sequences and by other receptors, in order to determine thespecificity and spectrum of their activity.

The transcriptional reporter assay, and most cell-based assays, are wellsuited for screening expression libraries for proteins for those thatmodulate FPRL2 polypeptide activity. The libraries can be, for example,cDNA libraries from natural sources, e.g., plants, animals, bacteria,etc., or they can be libraries expressing randomly or systematicallymutated variants of one or more polypeptides. Genomic libraries in viralvectors can also be used to express the mRNA content of one cell ortissue in the different libraries used for screening of FPRL2polypeptide.

h) Inositol Phosphates (IP) Measurement

Cells of the invention, for example, CHO-K1 cells, are labelled for 24hours with 10 μCi/ml [³H] inositol in inositol free DMEM containing 5%FCS, antibiotics, amphotericin, sodium pyruvate and 400 μg/ml G418.Cells are incubated for 2 h in Krebs-Ringer Hepes (KRH) buffer of thefollowing composition (124 mM NaCl, 5 mM KCl, 1.25 mM MgSO₄, 1.45 mMCaCl₂, 1.25 mM KH₂PO₄, 25 mM Hepes (pH:7.4) and 8 mM glucose). The cellsare then challenged with HBP polypeptide for 30 min. The incubation isstopped by the addition of an ice cold 3% perchloric acid solution. IPare extracted and separated on Dowex columns as previously described(25).

FPRL2 Polypeptide Assay

The invention provides for an assay for detecting the activity of areceptor of the invention in a sample. For example, FPRL2 polypeptideactivity can be measured in a sample comprising a cell or a cellmembrane that expresses FPRL2 polypeptide. As above, HBP polypeptide(SEQ ID NO: 18) is used as an example in this section. It should beunderstood that any HBP polypeptide as defined herein can be used inthese assays. The assay is performed by incubating the sample in thepresence or absence of HBP polypeptide and carrying out a secondmessenger assay, as described above. The results of the second messengerassay performed in the presence or absence of HBP polypeptide arecompared to determine if the FPRL2 polypeptide receptor is active. Anincrease of 10% or more in the detected level of a given secondmessenger, as defined herein, in the presence of HBP polypeptiderelative to the amount detected in an assay performed in the absence ofHBP polypeptide is indicative of FPRL2 polypeptide activity.

Any of the assays of receptor activity, including but not limited to theGTP-binding, GTPase, adenylate cyclase, cAMP, phospholipid-breakdown,diacylglycerol, inositol trisphosphate, arachidonic acid release (seebelow), PKC, kinase and transcriptional reporter assays, can be used todetermine the presence of an agent in a sample, e.g., a tissue sample,that affects the activity of the FPRL2 polypeptide receptor molecule. Todo so, FPRL2 polypeptide is assayed for activity in the presence andabsence of the sample or an extract of the sample. An increase in FPRL2polypeptide activity in the presence of the sample or extract relativeto the absence of the sample indicates that the sample contains anagonist of the receptor activity. A decrease in receptor activity in thepresence of HBP polypeptide or another agonist and the sample, relativeto receptor activity in the presence of HBP polypeptide alone, indicatesthat the sample contains an antagonist of FPRL2 polypeptide activity. Ifdesired, samples can then be fractionated and further tested to isolateor purify the agonist or antagonist. The amount of increase or decreasein measured activity necessary for a sample to be said to contain amodulator depends upon the type of assay used. Generally, a 10% orgreater change (increase or decrease) relative to an assay performed inthe absence of a sample indicates the presence of a modulator in thesample. One exception is the transcriptional reporter assay, in which atleast a two-fold increase or 10% decrease in signal is necessary for asample to be said to contain a modulator. It is preferred that anagonist stimulates at least 50%, and preferably 75% or 100% or more,e.g., 2-fold, 5-fold, 10-fold or greater receptor activation than withHBP polypeptide alone.

Other functional assays include, for example, microphysiometer orbiosensor assays (see Hafner, 2000, Biosens. Bioelectron. 15: 149-158,incorporated herein by reference). The intracellular level ofarachidonic acid can also be determined as described in Gijon et al.,2000, J. Biol. Chem., 275:20146-20156.

II. Diagnostic Assays Based Upon the Interaction of FPRL2 Polypeptideand HBP polypeptide:

Signalling through GPCRs is instrumental in the pathology of a largenumber of diseases and disorders. FPRL2 polypeptide, which is expressedin cells of the lymphocyte lineages, spleen, small intestine, lung,heart, can have a role in immune processes, cancer and associateddisorders or diseases.

The expression pattern of FPRL2 polypeptide and the knowledge withrespect to disorders generally mediated by GPCRs suggests that FPRL2polypeptide can be involved in disturbances of cell migration, cancer,development of tumours and tumour metastasis, inflammatory andneoplastic processes, wound and bone healing and dysfunction ofregulatory growth functions, obesity, anorexia, bulimia, acute heartfailure, hypotension, hypertension, urinary retention, osteoporosis,angina pectoris, restenosis, atherosclerosis, thrombosis and othercardiovascular diseases, autoimmune and, diseases characterized byexcessive smooth muscle cell proliferation, aneurysms, diseasescharacterized by loss of smooth muscle cells or reduced smooth musclecell proliferation, stroke, ischemia, ulcers, allergies, prostatichypertrophy, migraine, vomiting, psychotic and neurological disorders,including anxiety, schizophrenia, manic depression, depression,delirium, dementia and severe mental retardation, degenerative diseases,neurodegenerative diseases such as Alzheimer's disease or Parkinson'sdisease, and dyskinasias, such as Huntington's disease or Gilles de laTourett's syndrome and other related diseases including thrombosis andother cardiovascular diseases, autoimmune and inflammatory diseases suchas psoriasis, Eczeme, inflammatory and trophic diseases of skin,rheumatoid arthritis, scleroderma, lupus, polymyositis, dermatomysitis,Crohn's disease, inflammatory bowel disease (IBD), Irritable BowelSyndrome, Ulcerative Colitis, Asthma, Chronic Obstructive PulmonaryDisease, Allergic Rhinitis, Fibromyalgia, Organ Transplant Rejection,Graft versus host disease, Multiple Sclerosis, Acute, Ischemic Stroke,Infectious diseases, Hepatitis A, Hepatitis B, Hepatitis C, Sepsis,Septic shock, Chronic bronchitis, infections such as bacterial, fungal,protozoan and viral infections, such as infections caused by HIV1 andHIV2, and pain, cancer, anorexia, bulimia, asthma, acute heart failure,hypertension, urinary retention, osteoporosis, angina pectoris,myocardial infarction, ulcers, allergies, benign prostatic hypertrophy,and Type 1 Diabetes, Type 2 Diabetes, Osteoarthritis, DiabeticRetinopathy, Diabetic Nephropathy and fertility dysfunctions, foetaldevelopmental disorders

The interaction of FPRL2 polypeptide with HBP polypeptide can be used asthe basis of assays for the diagnosis or monitoring of diseases,disorders or processes involving FPRL2 polypeptide signalling.Diagnostic assays for FPRL2 polypeptide-related diseases or disorderscan have several different forms. First, diagnostic assays can measurethe amount of FPRL2 polypeptides, mRNA or ligand in a sample of tissue.Assays that measure the amount of mRNA encoding FPRL2 polypeptide alsofit into this category. Second, assays can evaluate the qualities of thereceptor or the ligand. For example, assays that determine whether anindividual expresses a mutant or variant form of FPRL2 polypeptide canbe used diagnostically. Third, assays that measure one or moreactivities of FPRL2 polypeptide can be used diagnostically.

A. Assays that Measure the Amount of FPRL2 Polypeptide

FPRL2 polypeptide levels can be measured and compared to standards inorder to determine whether an abnormal level of the receptor or itsligand is present in a sample, either of which indicate probabledysregulation of FPRL2 polypeptide signalling. Polypeptide levels aremeasured, for example, by immunohistochemistry using antibodies specificfor the polypeptide. A sample isolated from an individual suspected ofsuffering from a disease or disorder characterized by FPRL2 polypeptideactivity is contacted with an antibody for a FPRL2 polypeptide, andbinding of the antibody is measured as known in the art (e.g., bymeasurement of the activity of an enzyme conjugated to a secondaryantibody).

Another approach to the measurement of FPRL2 polypeptide levels usesflow cytometry analysis of cells from an affected tissue. Methods offlow cytometry, including the fluorescent labeling of antibodiesspecific for FPRL2 polypeptide, are well known in the art. Otherapproaches include radioimmunoassay or ELISA. Methods for each of theseare also well known in the art.

The amount of binding detected is compared to the binding in a sample ofsimilar tissue from a healthy individual, or from a site on the affectedindividual that is not so affected. An increase of 10% or more relativeto the standard is diagnostic for a disease or disorder characterized byFPRL2 polypeptide dysregulation.

FPRL2 polypeptide expression can also be measured by determining theamount of mRNA encoding the polypeptides in a sample of tissue. Levelsof mRNA can be measured by quantitative or semi-quantitative PCR.Methods of “quantitative” amplification are well known to those of skillin the art, and primer sequences for the amplification of FPRL2 nucleicacid are disclosed herein. A common method of quantitative PCR involvessimultaneously co-amplifying a known quantity of a control sequenceusing the same primers. This provides an internal standard that can beused to calibrate the PCR reaction. Detailed protocols for quantitativePCR are provided in PCR Protocols, A Guide to Methods and Applications,Innis et al., Academic Press, Inc. N.Y., (1990), which is incorporatedherein by reference. An increase of 10% or more in the amount of mRNAencoding FPRL2 polypeptide in a sample, relative to the amount expressedin a sample of like tissue from a healthy individual or in a sample oftissue from an unaffected location in an affected individual isdiagnostic for a disease or disorder characterized by dysregulation ofFPRL2 polypeptide signalling.

B. Qualitative Assays

Assays that evaluate whether or not a FPRL2 polypeptide or the mRNAencoding it are wild-type or not can be used diagnostically. In order todiagnose a disease or disorder characterized by FPRL2 polypeptidedysregulation in this manner, RNA isolated from a sample is used as atemplate for PCR amplification of FPRL2 polypeptide. The amplifiedsequences are then either directly sequenced using standard methods, orare first cloned into a vector, followed by sequencing. A difference inthe sequence that changes one or more encoded amino acids relative tothe sequence of wild-type FPRL2 polypeptide can be diagnostic of adisease or disorder characterized by dysregulation of FPRL2 polypeptidesignalling. It can be useful, when a change in coding sequence isidentified in a sample, to express the variant receptor or ligand andcompare its activity to that of wild type FPRL2 polypeptide. Among otherbenefits, this approach can provide novel mutants, includingconstitutively active and null mutants.

In addition to standard sequencing methods, amplified sequences can beassayed for the presence of specific mutations using, for example,hybridization of molecular beacons that discriminate between wild typeand variant sequences. Hybridization assays that discriminate on thebasis of changes as small as one nucleotide are well known in the art.Alternatively, any of a number of “minisequencing” assays can beperformed, including, those described, for example, in U.S. Pat. Nos.5,888,819, 6,004,744 and 6,013,431 (incorporated herein by reference).These assays and others known in the art can determine the presence, ina given sample, of a nucleic acid with a known polymorphism.

If desired, array or microarray-based methods can be used to analyze theexpression or the presence of mutation, in FPRL2 polypeptide sequences.Array-based methods for minisequencing and for quantitation of nucleicacid expression are well known in the art.

C. Functional Assays.

Diagnosis of a disease or disorder characterized by the dysregulation ofFPRL2 polypeptide signalling can also be performed using functionalassays. To do so, cell membranes or cell extracts prepared from a tissuesample are used in an assay of FPRL2 polypeptide activity as describedherein (e.g., ligand binding assays, the GTP-binding assay, GTPaseassay, adenylate cyclase assay, cAMP assay, arachidonic acid level,phospholipid breakdown, diacyl glycerol or inositol trisphosphateassays, PKC activation assay, or kinase assay). The activity detected iscompared to that in a standard sample taken from a healthy individual orfrom an unaffected site on the affected individual. As an alternative, asample or extract of a sample can be applied to cells expressing FPRL2polypeptide, followed by measurement of FPRL2 polypeptide signallingactivity relative to a standard sample. A difference of 10% or more inthe activity measured in any of these assays, relative to the activityof the standard, is diagnostic for a disease or disorder characterizedby dysregulation of FPRL2 polypeptide signalling.

Modulation of FPRL2 Polypeptide Activity in a Cell According to theInvention

The discovery of HBP polypeptide as a ligand of FPRL2 polypeptideprovides methods of modulating the activity of a FPRL2 polypeptidepolypeptide in a cell. FPRL2 polypeptide activity is modulated in a cellby delivering to that cell an agent that modulates the function of aFPRL2 polypeptide polypeptide. This modulation can be performed incultured cells as part of an assay for the identification of additionalmodulating agents, or, for example, in an animal, including a human.Agents include HBP polypeptide and other ligands as defined herein, aswell as additional modulators identified using the screening methodsdescribed herein including but not limited to any of the HBP polypeptideanalogues.

An agent can be delivered to a cell by adding it to culture medium. Theamount to deliver will vary with the identity of the agent and with thepurpose for which it is delivered. For example, in a culture assay toidentify antagonists of FPRL2 polypeptide activity, one will preferablyadd an amount of agent, e.g., HBP polypeptide that half-maximallyactivates the receptors (e.g., approximately EC₅₀), preferably withoutexceeding the dose required for receptor saturation. This dose can bedetermined by titrating the amount of HBP polypeptide to determine thepoint at which further addition of HBP polypeptide has no additionaleffect on FPRL2 polypeptide activity.

When a modulator of FPRL2 polypeptide activity is administered to ananimal for the treatment of a disease or disorder, the amountadministered can be adjusted by one of skill in the art on the basis ofthe desired outcome. Successful treatment is achieved when one or moremeasurable aspects of the pathology (e.g., tumor cell growth,accumulation of inflammatory cells) is changed by at least 10% relativeto the value for that aspect prior to treatment.

Candidate Modulators Useful According to the Invention

The invention provides for a compound that is a modulator of a receptorof the invention.

Preferably a candidate modulator is a HBP polypeptide (SEQ ID NO:18), aHBP polypeptide as defined herein above, a ligand as defined hereinabove or an agent, identified according to the invention.

The candidate compound can be a synthetic compound, or a mixture ofcompounds, or may be a natural product (e.g. a plant extract or culturesupernatant). A candidate compound according to the invention includesbut is not limited to a small molecule that can be synthesized, anatural extract, peptides, polypeptides, carbohydrates, lipids, anantibody or antigen-binding fragment thereof, nucleic acids, and a smallorganic molecules.

Candidate modulator compounds from large libraries of synthetic ornatural compounds can be screened. Numerous means are currently used forrandom and directed synthesis of saccharide, peptide, and nucleic acidbased compounds. Synthetic compound libraries are commercially availablefrom a number of companies including Maybridge Chemical Co. (Trevillet,Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates(Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemicallibrary is available from Aldrich (Milwaukee, Wis.). Combinatoriallibraries are available and can be prepared. Alternatively, libraries ofnatural compounds in the form of bacterial, fungal, plant and animalextracts are available from e.g., Pan Laboratories (Bothell, Wash.) orMycoSearch (NC), or are readily producible by methods well known in theart. Additionally, natural and synthetically produced libraries andcompounds are readily modified through conventional chemical, physical,and biochemical means.

Useful compounds may be found within numerous chemical classes. Usefulcompounds may be organic compounds, or small organic compounds. Smallorganic compounds have a molecular weight of more than 50 yet less thanabout 2,500 daltons, preferably less than about 750, more preferablyless than about 350 daltons. Exemplary classes include heterocycles,peptides, saccharides, steroids, and the like. The compounds may bemodified to enhance efficacy, stability, pharmaceutical compatibility,and the like. Structural identification of an agent may be used toidentify, generate, or screen additional agents. For example, wherepeptide agents are identified, they may be modified in a variety of waysto enhance their stability, such as using an unnatural amino acid, suchas a D-amino acid, particularly D-alanine, by functionalizing the aminoor carboxylic terminus, e.g. for the amino group, acylation oralkylation, and for the carboxyl group, esterification or amidification,or the like.

For primary screening, a useful concentration of a candidate compoundaccording to the invention is from about 10 nM to about 100 μM or more(i.e. 1 mM, 10 mM, 100 mM, or even 1M), but can also be 1 nM and higher,1 pM and higher, or 1 fM and higher. The primary screening concentrationwill be used as an upper limit, along with nine additionalconcentrations, wherein the additional concentrations are determined byreducing the primary screening concentration at half-log intervals (e.g.for 9 more concentrations) for secondary screens or for generatingconcentration curves.

Antibodies Useful According to the Invention

The invention provides for antibodies to FPRL2 polypeptide. Antibodiescan be made using standard protocols known in the art (See, for example,Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold SpringHarbor Press: 1988)). A mammal, such as a mouse, hamster, or rabbit canbe immunized with an immunogenic form of the peptide (e.g., FPRL2polypeptide or an antigenic fragment which is capable of eliciting anantibody response, or a fusion protein as described herein above).Immunogens for raising antibodies are prepared by mixing thepolypeptides (e.g., isolated recombinant polypeptides or syntheticpeptides) with adjuvants. Alternatively, FPRL2 polypeptides or peptidesare made as fusion proteins to larger immunogenic proteins. Polypeptidescan also be covalently linked to other larger immunogenic proteins, suchas keyhole limpet hemocyanin. Alternatively, plasmid or viral vectorsencoding FPRL2 polypeptide, or a fragment of these proteins, can be usedto express the polypeptides and generate an immune response in an animalas described in Costagliola et al., 2000, J. Clin. Invest. 105:803-811,which is incorporated herein by reference. In order to raise antibodies,immunogens are typically administered intradermally, subcutaneously, orintramuscularly to experimental animals such as rabbits, sheep, andmice. In addition to the antibodies discussed above, geneticallyengineered antibody derivatives can be made, such as single chainantibodies.

The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA, flow cytometry or otherimmunoassays can also be used with the immunogen as antigen to assessthe levels of antibodies. Antibody preparations can be simply serum froman immunized animal, or if desired, polyclonal antibodies can beisolated from the serum by, for example, affinity chromatography usingimmobilized immunogen.

To produce monoclonal antibodies, antibody-producing splenocytes can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with FPRL2 polypeptide,and monoclonal antibodies isolated from the media of a culturecomprising such hybridoma cells.

In addition, a functional fragment of an antibody, including fragment ofchimeric, humanized, primatized or single chain antibody, can also beproduced. Functional fragments of the foregoing antibodies retain atleast one binding function and/or modulation function of the full-lengthantibody from which they are derived. Preferred functional fragmentsretain an antigen-binding function of a corresponding full-lengthantibody (e.g., retain the ability to bind a human FPRL2). Particularlypreferred functional fragments retain the ability to inhibit or activateone or more functions characteristic of a FPRL2, such as a bindingactivity, a signaling activity, and/or stimulation of a cellularresponse. For example, in one embodiment, a functional fragment caninhibit or activate the interaction of FPRL2 with one or more of itsligands, and/or can inhibit or activate one or more receptor-mediatedfunctions.

For example, antibody fragments capable of binding to a human FPRL2receptor or portion thereof, including, but not limited to, scFv, Fv,Fab, Fab′ and F(ab′)² fragments are encompassed by the invention. Suchfragments can be produced by enzymatic cleavage or by recombinanttechniques, for example. For instance, papain or pepsin cleavage cangenerate Fab or F(ab′)² fragments, respectively. Antibodies can also beproduced in a variety of truncated forms using antibody genes in whichone or more stop codons has been introduced upstream of the natural stopsite. For example, a chimeric gene encoding a F(ab′)² heavy chainportion can be designed to include DNA sequences encoding the CH1 domainand hinge region of the heavy chain.

The sequence of an antibody obtainable according to a screening method(e.g. FPRL2 422F 2B9 1C11, FPRL2 422F 2G3 1A10) can be an homologoussequence (which may exist in other mammal species or specific groups ofhuman populations), where homology indicates sequence identity, means asequence which presents a high sequence identity (more than 80%, 85%,90%, 95% or 98% sequence identity) with the complete nucleotide or aminoacid sequence of an antibody or fragment thereof. A functional homologis characterized by the ability to bind FPRL2 as defined herein or bythe ability to inhibit or stimulate a signal in response in FPRL2, orboth.

Homologous sequences of an antibody sequence according to the inventionmay include an amino acid or nucleotide sequence encoding a similarsequence which exists in other animal species (rat, human, cat, dog,etc.) or in specific human population groups, but which are involved inthe same biochemical pathway.

Such homologous sequences may comprise additions, deletions orsubstitutions of one or more amino acids or nucleotides, which do notsubstantially alter the functional characteristics of the antibody orfragment thereof according to the invention. That is, homologs will haveat least 90% of the activity of an amino acid sequence of an antibody orfragment thereof and will bind, stimulate or inhibit FPRL2 specifically.

Such homologous sequences can also be nucleotide sequences of more than50, 100, 200, 300, 400, 600, 800 or 1000 nucleotides which are able tohybridize to the amino acid sequence of any antibody or fragment thereofunder stringent hybridisation conditions (such as the ones described bySAMBROOK et al., Molecular Cloning, Laboratory Manuel, Cold Spring,Harbor Laboratory press, New York). An example of “stringenthybridization conditions” is as follows: hybridize in 50% formamide,5×SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, 50 μg/ml sonicated salmon sperm DNA, 0.1% SDS and10% dextran sulfate at 42° C.; and wash at 42° C. (or higher, e.g., upto two degrees C. below the T_(m) of the perfect complement of the probesequence) in 0.2×SSC and 0.1% SDS.

High Throughput Screening Kit

A high throughput screening kit according to the invention comprises allthe necessary means and media for performing the detection of amodulator compound including an agonist, antagonist, inverse agonist orinhibitor to the receptor of the invention in the presence or absence ofHBP polypeptide, preferably at a concentration in the range of 1 nM to 1μM. The kit comprises materials to perform the following successivesteps. Recombinant cells of the invention, comprising and expressing thenucleotide sequence encoding the FPRL2 polypeptide receptor, are grownon a solid support, such as a microtiter plate, more preferably a 96well microtiter plate, according to methods well known to the personskilled in the art, especially as described in WO 00/02045. Modulatorcompounds according to the invention, at concentrations from about 1 nMto 1 μM or more, are added to the culture media of defined wells in thepresence or absence of an appropriate concentration of HBP polypeptide(preferably in the range of 1 nM to 1 μM).

Kits according to the invention can also comprise materials necessaryfor second messenger assays amenable to high throughput screeninganalysis, including but not limited to the measurement of intracellularlevels of cAMP, intracellular inositol phosphate, intracellulardiacylglycerol concentrations, arachinoid acid concentration or MAPkinase or tyrosine kinase activity (as described above). For example,the FPRL2 polypeptide activity, as measured in a cyclic AMP assay, isquantified by a radioimmunoassay as previously described (26). Resultsare compared to the baseline level of FPRL2 polypeptide activityobtained from recombinant cells according to the invention in thepresence of HBP polypeptide but in the absence of added modulatorcompound. Wells showing at least 2 fold, preferably 5 fold, morepreferably 10 fold and most preferably a 100 fold or more increase ordecrease in FPRL2 polypeptide activity as compared to the level ofactivity in the absence of modulator, are selected for further analysis.

Other Kits Useful According to the Invention

The invention provides for kits useful for screening for modulators ofFPRL2 polypeptide activity, as well as kits useful for diagnosis ofdiseases or disorders characterized by dysregulation of FPRL2polypeptide signalling. Kits useful according to the invention caninclude an isolated FPRL2 polypeptide (including a membrane- orcell-associated FPRL2 polypeptide, e.g., on isolated membranes, cellsexpressing FPRL2 polypeptide, or on an SPR chip). A kit can alsocomprise an antibody specific for FPRL2 polypeptide. Alternatively, orin addition, a kit can contain cells transformed to express FPRL2polypeptide. In a further embodiment, a kit according to the inventioncan contain a polynucleotide encoding a FPRL2 polypeptide. In a stillfurther embodiment, a kit according to the invention may comprise thespecific primers useful for amplification of FPRL2 polypeptide asdescribed below. All kits according to the invention will comprise thestated items or combinations of items and packaging materials therefor.Kits will also include instructions for use.

Transgenic Animals

Transgenic mice provide a useful tool for genetic and developmentalbiology studies and for the determination of the function of a novelsequence. According to the method of conventional transgenesis,additional copies of normal or modified genes are injected into the malepronucleus of the zygote and become integrated into the genomic DNA ofthe recipient mouse. The transgene is transmitted in a Mendelian mannerin established transgenic strains. Constructs useful for creatingtransgenic animals comprise genes under the control of either theirnormal promoters or an inducible promoter, reporter genes under thecontrol of promoters to be analyzed with respect to their patterns oftissue expression and regulation, and constructs containing dominantmutations, mutant promoters, and artificial fusion genes to be studiedwith regard to their specific developmental outcome. Typically, DNAfragments on the order of 10 kilobases or less are used to construct atransgenic animal (Reeves, 1998, New. Anat., 253:19). Transgenic animalscan be created with a construct comprising a candidate gene containingone or more polymorphisms according to the invention. Alternatively, atransgenic animal expressing a candidate gene containing a singlepolymorphism can be crossed to a second transgenic animal expressing acandidate gene containing a different polymorphism and the combinedeffects of the two polymorphisms can be studied in the offspringanimals.

Other Transgenic Animals

The invention provides for transgenic animals that include but are notlimited to transgenic mice, rabbits, rats, pigs, sheep, horses, cows,goats, etc. A protocol for the production of a transgenic pig can befound in White and Yannoutsos, Current Topics in Complement Research:64^(th) Forum in Immunology, pp. 88-94; U.S. Pat. No. 5,523,226; U.S.Pat. No. 5,573,933: PCT Application WO93/25071; and PCT ApplicationWO95/04744. A protocol for the production of a transgenic mouse can befound in U.S. Pat. No. 5,530,177. A protocol for the production of atransgenic rat can be found in Bader and Ganten, Clinical andExperimental Pharmacology and Physiology, Supp. 3:S81-S87, 1996. Aprotocol for the production of a transgenic cow can be found inTransgenic Animal Technology, A Handbook, 1994, ed., Carl A. Pinkert,Academic Press, Inc. A protocol for the production of a transgenicrabbit can be found in Hammer et al., Nature 315:680-683, 1985 andTaylor and Fan, Frontiers in Bioscience 2:d298-308, 1997.

Knock Out Animals

i. Standard

Knock out animals are produced by the method of creating gene deletionswith homologous recombination. This technique is based on thedevelopment of embryonic stem (ES) cells that are derived from embryos,are maintained in culture and have the capacity to participate in thedevelopment of every tissue in the mouse when introduced into a hostblastocyst. A knock out animal is produced by directing homologousrecombination to a specific target gene in the ES cells, therebyproducing a null allele of the gene. The potential phenotypicconsequences of this null allele (either in heterozygous or homozygousoffspring) can be analyzed (Reeves, supra).

ii. In Vivo Tissue Specific Knock Out in Mice Using Cre-lox.

The method of targeted homologous recombination has been improved by thedevelopment of a system for site-specific recombination based on thebacteriophage P1 site specific recombinase Cre. The Cre-loxPsite-specific DNA recombinase from bacteriophage P1 is used intransgenic mouse assays in order to create gene knockouts restricted todefined tissues or developmental stages. Regionally restricted geneticdeletion, as opposed to global gene knockout, has the advantage that aphenotype can be attributed to a particular cell/tissue (Marth, 1996,Clin. Invest. 97: 1999). In the Cre-loxP system one transgenic mousestrain is engineered such that loxP sites flank one or more exons of thegene of interest. Homozygotes for this so called ‘floxed gene’ arecrossed with a second transgenic mouse that expresses the Cre gene undercontrol of a cell/tissue type transcriptional promoter. Cre protein thenexcises DNA between loxP recognition sequences and effectively removestarget gene function (Sauer, 1998, Methods, 14:381). There are now manyin vivo examples of this method, including the inducible inactivation ofmammary tissue specific genes (Wagner et al., 1997, Nucleic Acids Res.,25:4323).

iii. Bac Rescue of Knock Out Phenotype

In order to verify that a particular genetic polymorphism/mutation isresponsible for altered protein function in vivo one can “rescue” thealtered protein function by introducing a wild-type copy of the gene inquestion. In vivo complementation with bacterial artificial chromosome(BAC) clones expressed in transgenic mice can be used for thesepurposes. This method has been used for the identification of the mousecircadian Clock gene (Antoch et al., 1997, Cell 89: 655).

Materials

Trypsin was from Flow Laboratories (Bioggio, Switzerland). Culturemedia, G418, fetal bovine serum (FBS), restriction enzymes, Pfu DNAPolymerase was purchased from Stratagene and Taq DNA polymerases werepurchased from Eurogentec. (Liege, Belgium). The radioactive productmyo-D-[2-³H]inositol (17.7 Ci/mmol) was from Amersham (Ghent, Belgium).Dowex AG1X8 (formate form) was from Bio-Rad Laboratories (Richmond,Calif.). ATP was obtained from Sigma Chemical Co. (St. Louis, Mo.).Forskolin was purchased from Calbiochem (Bierges, Belgium). Rolipram wasa gift from the Laboratories Jacques Logeais (Trappes, France). PCDNA3is an expression vector obtained from Invitorgen.

Dosage and Mode of Administration

By way of example, a patient can be treated as follows by theadministration of a modulator of FPRL2 polypeptide (for example, anagonist, antagonist or inhibitor of FPRL2 polypeptide, of theinvention). A modulator of FPRL2 polypeptide of the invention can beadministered to the patient, preferably in a biologically compatiblesolution or a pharmaceutically acceptable delivery vehicle, byingestion, injection, inhalation or any number of other methods. Thedosages administered will vary from patient to patient; a“therapeutically effective dose” can be determined, for example, by thelevel of enhancement of function (e.g., as determined in a secondmessenger assay described herein). Monitoring HBP polypeptide bindingwill also enable one skilled in the art to select and adjust the dosagesadministered. The dosage of a modulator of FPRL2 polypeptide of theinvention may be repeated daily, weekly, monthly, yearly, or asconsidered appropriate by the treating physician.

In one embodiment, a patient can be treated to modulate the signallingactivity of a FPRL2 polypeptide receptor by administering to a patient asublethal dose of an agent which inhibits or promotes the signallingactivity of FPRL2 polypeptide. A sublethal dose according to theinvention, refers to a dose of an agent for inhibiting or stimulating aFPRL2 polypeptide signalling activity which is at or below the LD50 forthe particular agent. In one embodiment, the dose of an agent whichinhibits the signalling activity of FPRL2 polypeptide is between 1 fMand 1 M, preferably between 1 pM and 1 mM, and more preferably between 1nM and 1 μM. In one embodiment, an agent useful for the modulation ofFPRL2 polypeptide signalling may be an antibody which specifically bindsto the ligand binding site of FPRL2 polypeptide. An amount of anti-FPRL2polypeptide antibody needed to achieve a dosage useful for themodulation of FPRL2 polypeptide signalling will depend upon the level ofexpression of FPRL2 polypeptide, localization of receptor expression,and general state of the patient's own immune system, but generallyrange from 0.0005 to 5.0 mg of anti-FPRL2 polypeptide antibody orbinding protein thereof per kilogram of body weight, with doses of 0.05to 2.0 mg/kg/dose being more commonly used.

Pharmaceutical Compositions

The invention provides for compositions comprising a FPRL2 polypeptidemodulator according to the invention admixed with a physiologicallycompatible carrier. As used herein, “physiologically compatible carrier”refers to a physiologically acceptable diluent such as water, phosphatebuffered saline, or saline, and further may include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminium phosphate,aluminium hydroxide, or alum are materials well known in the art.

The invention also provides for pharmaceutical compositions. In additionto the active ingredients, these pharmaceutical compositions may containsuitable pharmaceutically acceptable carrier preparations which can beused pharmaceutically.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethyl cellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer' solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

For nasal administration, penetrants appropriate to the particularbarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner known in the art, e.g. by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc . . . Saltstend to be more soluble in aqueous or other protonic solvents that arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2%sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combinedwith buffer prior to use.

After pharmaceutical compositions comprising a compound of the inventionformulated in a acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition with information including amount, frequency andmethod of administration.

EXAMPLES Example 1 Cloning of Human FPRL2 Receptor

Human FPRL2 was cloned as follows: Oligonucleotides were synthesized,corresponding to the sequence of FPRL2. Oligonucleotide AS-204 had theforward sequence: 5′-ACCGGAATTCACCATGGAAACCAACTTCTCC-3′ (SEQ ID NO: 14),and hybridized on the translation initiation codon of FPRL2.Oligonucleotide AS-416 had the sequence:5′-ATCATCTAGAACGCAGGGTAGAAAGAGACAG-3′ (SEQ ID NO: 15), and wascomplementary to a sequence located downstream of the translation stopcodon of FPRL2. A PCR was performed, using human genomic DNA astemplate, and using oligonucleotides AS-204 and AS-416 as primers, withthe following conditions:

PCR enzyme: Pfu DNA Polymerase (Stratagene).

Buffer supplied by the Stratagene with the enzyme, and added with 2.5%(v/v) of DMSO Cycles were as follow:

Temperature (° C.) Time (min) 1x 94 5′ 3x 94 1′ 48 1′ 72 2′ 30x  94 1′60 1′ 72 2′ 1x 72 10′ 

A PCR product of the expected size (1.1 kb) was obtained. This productwas cloned in the EcoRI and XbaI sites of an expression vector (PCDNA3),using the EcoRI site introduced by the AS-204 oligonucleotide, and theXbaI site introduced by the AS-416 oligonucleotide and sequenced on bothstrand (FIG. 1).

Example 2 Tissue Distribution of FPRL2

Tissue distribution of human FPRL2-Reverse transcription-polymerasechain reaction (RT-PCR) experiments were carried out using a panel ofpoly(A)+ RNA (spinal cord, thymus, pancreas, uterus, placenta, stomach,lung, spleen, testis, brain, heart, kidney, skeletal muscle, fetalliver, fetal brain, adrenal gland, bone marrow) and total RNA(pituitary, small intestine, liver, ovary, fetal aorta, adipose,monocytes, lymph node, T lymphocytes, B lymphocytes, PBMC, PMN, PBL).The FPRL2 primers were 5′-CGCACAGTCAACACCATCTG-3′ (forward) (SEQ ID NO:16) and 5′-AGCTGTTAAAAAAGGCCAAG-3′ (reverse) (SEQ ID NO: 17), with anexpected product size of 717 bp (FIG. 2). Approximately 50 ng ofPoly(A)+ RNA or 500 ng of total RNA was reverse transcribed withSuperscript II (Invitrogen) and used for PCR. PCR was performed usingthe Taq polymerase under the following conditions: denaturation at 94°C. for 5 min, 30 cycles at 94° C. for 1 min, 56° C. for 1 min 30 s, and72° C. for 45 s. Aliquots (10 μl) of the PCRs were analyzed by 1%agarose gel electrophoresis.

Example 3 Purification of the Natural Ligand of FPRL2 and Identificationof a Fragment of HBP

1. Homogenate

The purification was performed from 350 g of porcine spleen. The freshorgan was shopped and frozen in liquid nitrogen. Frozen organ was thenbrought to 4° C. in a solution of 20% acetonitrile (ACN) in water, in a¼ organ/liquid proportion. The mixture was mixed, then reduced to anhomogenate with an Ultraturax.

After homogeneization, the mixture was centrifuged at 10000 g for 30minutes at 4° C.

The supernatant was frozen in liquid nitrogen and stored at −80° C.

2. First Step

Aliquots of 200 ml, corresponding to 50 g of organ, were diluted 4 timesin water 0.1% trifluoroacetic acid (TFA) to reach a concentration of 5%ACN. 800 ml were then loaded on a Poros column 4.6×150 mm at 5 ml/min.The column was submitted to a gradient of ACN (supplemented with 0.1%TFA) from 5 to 70% at 6%/minute. Fractions of 1.25 ml were collected andtested for functional activity in an aequorin assay. Two regions ofactivity were detected on the HPLC profile and the correspondingfractions were conserved.

3. Second Step

The fractions corresponding to the two regions of activity were pooled.The pool was diluted 4 times in water 0.1% TFA and loaded on a C18column 4.6×250 mm at 1 ml/min. The column was submitted to gradient ofACN in the presence of 0.1% TFA at a rate of 1%/min between 30 and 50%.Fractions of 1 ml were collected and tested for functional activity. Tworegions of activity were detected. From this step on, the two regionswere treated separately.

4. Third Step

The fractions corresponding to the second region (higher percentage ofacetonitrile) from 2 runs C18 were pooled and concentrated in a speedvacto a final volume of 50 μl. This was then diluted 3 times in a mixturewater/30% ACN/0.1% TFA. This final volume was loaded on a size-exclusioncolumn Superdex peptide PE 7.5×300 mm (Pharmacia Biotech) equilibratedwith the dilution buffer. The run was conducted at 0.5 ml/min andfractions of 0.25 ml were collected and tested for functional activity.

5. Fourth Step

The active fractions from one step 4 were pooled and diluted 4 times inwater 0.1% TFA. The final volume (2 ml) was loaded at 0.2 ml/min on a C4column 2.1×250 mm. The column was submitted to a gradient of ACN in thepresence of 0.1% TFA at a rate of 0.3%/min between 30 and 50%. Thefractions were collected manually according to the absorbance profileand tested for functional activity. This step was repeated 3 times. Thepurity of the final active fraction was checked by loading an aliquot ofit on a C18 column 1×250 mm. This fraction was then dried, resuspendedin 20 mM ammonium bicarbonate, boiled, digested by trypsin (50 ng)overnight and finally analysed on a MALDI-Q-TOF mass spectrometer.Direct monoisotopic mass fingerprinting allowed to identify thefollowing peptide: Acetyl-MLGMIKNSLFGSVETWPWQVL (Sequence ID NO: 18)which corresponds to the 21 first amino acids of the porcine HBP (FIG.3). The 21 first amino acid of human HBP are 100% identical to the 21first amino acid of porcine HBP.

Example 4 Functional Assay for FPRL2

FPRL2 expressing clones have been obtained by transfection of CHO-K1cells to coexpressing mitochondrial apoaequorin and Galpha16, limitingdilution and selection by northern blotting. Positive clones were usedfor screening with porcine spleen extracts prepared as described above.A functional assay based on the luminescence of mitochondrial aequorinintracellular Ca²⁺ release (Stables et al., 1997, Anal. Biochem.252:115-126; incorporated herein by reference) was performed asdescribed (Detheux et al., 2000, J. Exp. Med., 192 1501-1508;incorporated herein by reference). Briefly, cells were collected fromplates in PBS containing 5 mM EDTA, pelleted and resuspended at 5×10⁶cells/ml in DMEM-F12 medium. Cells were incubated with 5 μMCoelenterazine H (Molecular Probes) for 4 hours at room temperature.Cells were then washed in DMEM-F12 medium and resuspended at aconcentration of 0.5×10⁶ cells/ml. Cells were then mixed with testagonist peptides or plates containing tissue extracts and the lightemission was recorded for 30 sec using a Microlumat luminometer (PerlinElmer). Results are expressed as Relative Light Units (RLU).

Example 5 Activation of Cells Expressing FPRL2 by N-Terminal Peptide ofHBP

In order to investigate the potential effect of the N-terminal domain ofHBP, we purified the natural peptide from the active spleen fraction(this peptide has the sequence shown in SEQ ID NO: 18), and tested itsability to trigger intracellular calcium release in a cell linecoexpressing the FPRL2 receptor and apoaequorin. We have used theaequorin assay as previously described in Detheux et al. (2000 J. Exp.Med. 192, 1501-1508). As shown in FIG. 4, the peptide of 21 amino acidscorresponding to the N-terminal end of porcine HBP was able to activatethe FPRL2 at nanomolar concentration (mean EC₅₀ of 3.6 nM).

In order to investigate whether the acetyl group, located at the NH2 endof the peptide shown by SEQ ID NO: 18, modulates the activity of thepeptide or not, we synthesized this peptide of 21 amino acids with andwithout acetyl group at its NH2 end. As shown in FIG. 5 the acetyl groupdoes not modify the activity of the peptide on FPRL2 receptor (mean EC₅₀of 21 nM for acetylated peptide and EC₅₀ of 36 nM for non acetylatedpeptide).

Example 6 N-Terminal Peptide of HBP Activates Specifically the FPRL2Receptor

In order to investigate the specificity of the above-mentioned peptideon FPRL2 receptor, we tested the activity of this peptide on two othermembers of FPR family: FPR receptor and FPRL1 receptor. As shown in FIG.6, the peptide of 21 amino acids (SEQ ID NO: 18) was able to activatethe FPRL2 receptor at nanomolar concentration (mean EC₅₀ of 8.8 and 8.2nM, notice: in this experiment two different clones expressing FPRL2were tested) but was not able to activate FPR and activated FPRL1 onlyat high concentration (mean EC₅₀ of 584 nM).

Example 7 FPRL2 is a Gi Coupled Receptor

The cAMP concentrations were determined using a HTRF kit, according tomanufacturer specifications (HTRF kit, Cis bio International, cat n^(o)62AM2PEC).

Cells in mid-log phase, grown in media without antibiotics for 18 hoursprior to the experiment, are detached by gentle flushing with PBS-EDTA,recovered by centrifugation and resuspended in KRH-IBMX (1 mM) at theconcentration of 4.2×10⁵ cells/ml.

For the agonist assay, 6 μl/well of Forskolin 4× (10 μM finalconcentration) and 6 μl of increasing amounts of tested agonist (4×)(peptide 2478: which corresponds to the peptide disclosed in SEQ ID NO:18) or Forskolin 4× (10 μM final concentration) for the FK control aredispensed in 96 well plate (Costar, cat n^(o): 3694). 12 μl/well of cellsuspension (5000 cells/well) are added onto each well and incubated for30 min at room temperature.

The reaction was stopped by successive addition of 12 μl of cAMP-XL665and 12 μl anti-cAMP cryptate diluted in Lysis buffer. The plate wasincubated for 60 min. at room temperature and read on Rubystar (BMG).Results are calculated from the 665 nm/620 nm ratio and expressed inDelta F (%). A calibration curve is obtained by plotting deltaF % versuscAMP concentrations. Delta F % obtained from samples can be reported onthe calibration curve to deduce respective cAMP concentrations (nM)produced by each sample.

The natural coupling properties and the intracellular signaling pathwaysactivated by FPRL2, upon stimulation by SEQ ID NO: 18 were investigatedin CHO-K1 cells expressing the human receptor and the aequorin. We firstdemonstrated that the FPRL2 receptor coupled negatively to adenylatecyclase in presence of forskolin (FIG. 7), while being unable to promoteaccumulation of cAMP in the absence of forskolin (not shown).

The peptide shown by SEQ ID NO: 18 is able to decrease the amount ofcAMP produced in cells expressing the FPRL2 receptor in a dose-dependentmanner and displays an agonist activity on FPRL2 with an EC₅₀ of6.5+/−2.4 nM.

Example 8 Identification, Synthesis, and Characterization of Human FPRL2Receptor and Ligand

A) Material and Methods

Expression of Human FPRL2, FPRL1 and FPR.

The human coding sequences (accession numbers AC005946, M84562 andM60626, respectively) were amplified by PCR from human genomic DNA,cloned into the pcDNA3 (Invitrogen) and pEFIN3 (Euroscreen) vectors andsequenced. The pEFIN3 constructs were transfected, using Fugene 6, intoCHO-K1 cells, expressing or not G_(α16) and apoaequorin. G418-resistantclones were characterized for receptor expression by Northern blotting.A functional assay based on the luminescence of mitochondrial aequorinwas performed as described (42). Results were expressed as relativelight units (RLU) or as the percentage of the response to 20 μM ATP.

Purification of Bioactive Peptides.

Frozen porcine spleen (350 g) was homogenized in four volumes ofice-cold 20% CH₃CN in water. The homogenate was centrifuged at 10,000 gfor 30 minutes at 4° C. and snap-frozen in liquid nitrogen. Aliquots of200 ml of supernatant were diluted four-fold in 0.1% trifluoroaceticacid (TFA) and loaded on a Poros R2 beads 4.6×150 mm column (AppliedBiosystems) at 5 ml/min. A 5-70% CH₃CN gradient (6%/min) in 0.1% TFA wasapplied, and 1.25 ml fractions were collected and tested for functionalactivity on FPRL2-expressing CHO-K1 cells in an aequorin assay. Tworegions of activity (A1 and A2) were detected on the HPLC profile. Thecorresponding fractions were pooled, diluted four-fold in 0.1% TFA andloaded at 1 ml/min on a C18 4.6×250 mm column (Vydac), which wassubmitted to a 30-50% CH₃CN gradient in 0.1% TFA. Two regions ofactivity were detected and subsequently treated separately. The 1 mlfractions corresponding to the first (A1, lower CH₃CN concentration) andthe second (A2, higher CH₃CN concentration) regions from two runs werevacuum-concentrated to 50 μl. A1 and A2 were diluted three-fold inrespectively 30% CH₃CN/0.05% TFA and 30% CH₃CN/0.1% TFA, and loaded onsize-exclusion columns (SEC) (A1: TSK-gel Alpha-4000 7.8×300 mm, TosohBiosep; A2: Superdex peptide PE 7.5×300 mm, Amersham Pharmacia Biotech)submitted to a 0.5 ml/min flow rate of dilution medium. The active 0.25ml fractions from one SEC were diluted four-fold in 0.1% TFA and loadedat 0.2 ml/min on a C4 2.1×250 mm column (Vydac), which was submitted toa 25-45% (A1) or 30-50% (A2) CH₃CN gradient at 0.3%/min in 0.1% TFA. Thefractions were collected manually according to the absorbance profile.For A1, the active fractions from one run were pooled, diluted five-foldin 0.1% TFA, and loaded at 0.05 ml/min on a C18 1×250 mm column (Vydac),which was submitted to a 23-50% CH₃CN gradient at 0.45%/min in 0.1% TFA.The fractions were collected manually. For A2, the purity of the finalactive fraction was checked by loading an aliquot on a C18 1×250 mmcolumn. The purification was repeated three times, with differentprotocols (i.e. the SEC step was replaced by a reverse-phase step on aC18 2.1×250 mm column submitted to a CH₃CN gradient in 0.1% H₃PO₄ asion-pairing agent). The protein concentration in active fractions wasdetermined following SDS/PAGE, by comparison with aprotinin and lysozymestandards following silver staining.

Mass Spectrometry Analysis.

The active fractions were vacuum-dried, resuspended in 20 mM ammoniumbicarbonate, heated to 100° C. for 5 min, digested by trypsin (50 ng)overnight or left intact, and purified by solid-phase extraction (C18ZipTip, Millipore). The peptides were eluted in 1.5 μl of 70% CH₃CN/0.1%TFA onto a metallic MALDI target, dried, and then mixed with 1.5 μl ofmatrix mix (2 mg/ml 2,5-dihydroxybenzoic acid and 10 mg/mlα-cyano-4-hydroxycinnamic acid, 2 mM fucose, 5 mM ammonium acetate).Mass spectrometry analysis was performed on a Q-TOF Ultima Global massspectrometer equipped with a MALDI source (Micromass), and calibratedusing the monoisotopic masses of tryptic and chymotryptic peptides frombovine serum albumin. Ionization was achieved using a nitrogen laser(337 nm beam, 10 Hz) and acquisitions were performed in a V modereflectron position. Microsequencing was performed by argon-inducedfragmentation after selection of the parent ion.

Synthetic Peptides.

Acetylated or non-acetylated MLGMIKNSLFGSVETWPWQVL(SEQ ID NO: 37)(HBP[1-21], F2L in this Example 8), NSLFGSVETWPWQVL (SEQ ID NO: 20)(F2L[7-21]), WKYMVM (SEQ ID NO: 21), and MLWRRKIGPQMTLSHAAG (SEQ ID NO:22)(SHAAG peptide derived from CCL23 N-terminus) were synthesizedlocally by using the solid phase Fmoc strategy (43) or custom made byEurogentec. WKYMVM (SEQ ID NO: 21)and WKYMVm (SEQ ID NO: 23) werepurchased from Phoenix Pharmaceuticals and FMLP from Neosystem.Monoisotopic masses and sequences of all peptides were verified by massspectrometry. F2L and WKYMVM (SEQ ID NO: 21) from different originsdisplayed the same properties. At high concentrations, HBP-derivedpeptides were dissolved in DMSO and heated at 50° C. for 10 mm, due totheir high hydrophobicity. Intermediate dilutions were made in 50%CH₃CN, and were further diluted 40-fold in assay buffer to reach workingconcentration.

Quantitative RT-PCR

For the quantitative PCR, FPRL2 transcripts were detected by RT-PCR incDNA from human blood cell populations obtained commercially (Clontech)or prepared locally as described (44). Primers were5′-CTGGCCACACCGTTCTGT-3′(SEQ ID NO: 24) as forward,5′-GGCCATGGTAATGAACACGTT-3′(SEQ ID NO: 25) as reverse for FPRL2.Amplification of GAPDH transcripts was performed as a control of thequality of cDNA (not shown). FPRL2 transcripts were detected byquantitative RT-PCR (TaqMan) in total or polyA+RNA samples from humantissues obtained commercially (Clontech and Ambion) or prepared locally(DCs). Primers were 5′-TTACCATGGCCAAGGTCTTTCT-3′(SEQ ID NO: 26) asforward, 5′-GCAGACTGTGATGATGGACATAGG-3′(SEQ ID NO: 27) as reverse and5′FAM-TCCTCCACTTCATTATTGGCTTCAGCGT-3′DABSYL (SEQ ID NO: 28) as probe forFPRL2, and 5′-GAAGGTGAAGGTCGGAGTC-3′(SEQ ID NO: 29) as forward,5′-GAAGATGGTGATGGGATTTC-3′(SEQ ID NO: 30) as reverse and5′FAM-CAAGCTTCCCGTTCTCAGCC-3′DABSYL (SEQ ID NO: 31) as probe for thereference housekeeping gene (GAPDH). Primers were used at 900 nM andprobes at 200 nM. Standard curves were run systematically for the twogenes, and the transcript copy number of FPRL2 was normalized to theGAPDH transcript copy number for each sample.

Monoclonal Antibodies and Flow Cytometry

Antibodies were generated by injecting BALB/c mice with 100 μgpcDNA3-FPRL2 as described (45). Sera were tested by FACS on theCH0-K1-FPRL2 cell line, and immune mice were used to generate monoclonalantibodies by standard hybridoma technology, using the NSO myeloma cellline. The Ig class of selected hybridomas was determined with a mousemAb isotyping kit (IsoStrip, Boehringer Mannheim). The antibodies weretested using flow cytometry, performed using anti-FPRL2 antibodies orcontrol IgG2a at 1 μg/ml (for CHO-K1 cells) or 5 μg/ml (for primarycells) in PBS containing 0.1% BSA, 0.1% sodium azide, andFITC-conjugated γ-chain-specific goat anti-mouse IgG (Sigma) assecondary antibody. Fluorescence of 10,000 cells was assayed using aFACScan flow cytofluorimeter (Beckton Dickinson). Intracytoplasmicstaining was realised using Cytoperm/Cytowash (Becton Dickinson)according to manufacturer's instructions.

Intracellular Cascade Assays

The cAMP concentrations were determined using a homogeneoustime-resolved fluorescence (HTRF) kit (Cis Bio International). Briefly,cells were detached, resuspended in Krebs Ringer Hepes buffer containing1 mM 3-Isobutyl-1-methylxanthine, and submitted to 10 μM forskolin,alone or together with increasing concentrations of agonists for 30 minat room temperature. The reaction was stopped by the successive additionof cAMP-XL665 and anti-cAMP cryptate diluted in lysis buffer. The plateswere incubated for 60 min at room temperature and read on a Rubystarfluorimeter (BMG). Results were calculated from the 665 nm/620 nm ratioand expressed in delta F (%). A calibration curve was obtained byplotting delta F % versus cAMP concentrations. ERK1/2 activation wasassayed by Western blotting, using an anti-phospho-p42/44 monoclonalantibody (E10, Cell Signaling Technology) as described (46). Theaequorin-based assay was performed with or without overnightpretreatment with 100 ng/ml Pertussis toxin. It was shown that suchPertussis toxin pretreatment did not inhibit the functional response toATP in these cells. For FPRL2 polymorphism analysis, HEK cells weretransiently transfected with empty and wild-type or Asp338H isFPRL2-containing pcdna vector using calcium phosphate method. Cells wererecovered 48 hours later and used for FACS or cAMP experiments.

Binding Assays

A modified F2L peptide, bearing a carboxy-terminal tyrosine, was shownto display a potency similar to that of wild-type F2L in the aequorineassay. 5 μg of peptide was labeled with 2 mCi of¹²⁵ I using the Jodogenmethod. Following separation of unbound ¹²⁵ I, the resulting specificactivity of the peptide was estimated to 900 Ci per mmole. [¹²⁵]-WKYMVm(SEQ ID NO: 23) (2200 Ci/mmole) was purchased from Perkin Elmer LifeSciences. FPRL2, FPRL1 and FPR-expressing CHO-K1 cells were plated in 24wells plates (200,000 cells per well for FPRL2, and 100,000 cells perwell for the two other receptors). The next day, the cells were washedtwice with a KRH buffer containing 280 mM saccharose and, for FPRL1 andFPR, 0.1 % NaN₃. For saturation binding assays, cells were incubatedwith various amounts of F2L-[¹²⁵ I]Tyr and non-specific binding wasdetermined by using 1 μM F2L as competitor. For competition bindingassays, cells were incubated with 100,000 cpm of F2L-[¹²⁵ I]Tyr or10,000 cpm of [¹²⁵ I]-WKYMVm (SEQ ID NO: 23) and various amounts of F2Lor other peptides as competitors, in KRH buffer supplemented with 5 %BSA, for 90 mm at room temperature. Cells were washed twice with icecold buffer, total radioactivity was recovered with 1 M NaGH and countedin a gamma counter for 2 mm

Chemotaxis and Ca2+ Mobilization Assays on Primary Cells

Monocyte-derived DCs were generated either from the adherent fraction ofPBMCs cultured with GM-CSF (800 U/ml) and 11-4 (500 U/ml), or fromPercoll-purified monocytes cultured with GM-CSF (50 ng/ml) and IL-13 (20ng/ml), for 5 to 7 days. For Ca²⁺ mobilization assay, monocytes wereobtained by negative selection with the Monocyte Isolation Kit II(Miltenyi Biotec). Cell migration in response to F2L and FMLP and Mip1alpha used as controls was evaluated by using a 48-well microchemotaxischamber technique as described (48). For Ca²⁺ mobilization assays,monocyte-derived DCs or monocytes (5×10⁵ cells/ml in HBSS without phenolred but containing 0.1% BSA and 1 mM Probenecid (Sigma)) were loadedwith 4 μM Fluo 4 (Molecular Probes) for 1 h at 20° C. in the dark. Theloaded cells were washed twice, resuspended at 1 to 2×10⁶ cells/ml and50 μl of cell suspension was distributed per well of a 96 well plate(Viewplate, Packard Bioscience). Reading was performed in a Fluostarfluorimeter (BMG) at 25° C.: 50 μl of ligand-containing medium wasinjected, and the fluorescence at 520 nm was recorded every second for 1to 3 min. Each condition was performed in triplicate, the meanfluorescence for each time point was calculated, and the curves werenormalized by subtracting the mean value of the five measurementspreceding the injection.

B) Results

Isolation and Identification of the F2L Peptide (SEQ ID No 18) as anEndogenous Ligand of FPRL2.

We developed, as a screening assay, CHO-K1 cell lines coexpressing humanFPRL2, apoaequorin and Galpha 16. This allowed to test fractions fromhuman lymphoid organ extracts, conditioned media of leukocytepopulations, and inflammatory fluids. A biological activity, specificfor FPRL2-expressing cell lines, was detected in fractions resultingfrom the reverse phase HPLC fractionation of extracts from human spleen(data not shown). For practical reasons, we tested fractions fromporcine spleen prepared in a similar way, and identified two regions ofthe profile containing specific activities for FPRL2 (activities A1 andA2, FIG. 9). Starting from 350 g of porcine spleen, these two activitieswere purified to homogeneity by five (A1) or four (A2) successive HPLCsteps, the first two being common (FIG. 9). The sensitivity of bothactivities to proteinase K suggested a peptidic nature (not shown). Themolecular mass of the active compounds was estimated by size-exclusionchromatography to about 6 kD for activity A1, and 3 kD for activity A2.From the absorbance of the peaks and the biological activitiesassociated with them, the compound present in peak A1 appeared moreabundant but less active than that of peak A2. The two fractions wereanalyzed by SDS/PAGE and silver staining, in order to quantifyapproximately the active peptides by comparison with known amounts ofaprotinin and lysozyme (FIGS. 10D and F). A concentration-action curveperformed on the same fractions in the aequorin-based functional assayallowed to estimate an EC50 of 2.32±1.84 nM (n=2) for A2, and of 200±54nM (n=4) for A1 (FIGS. 10G and E, respectively). Both peptides wereanalyzed by mass spectrometry, either without (A2) or after trypticdigestion (A1 and A2) (FIGS. 10A and B). For A2, analysis of theundigested peptide permitted to identify the entire microsequence asmatching the first 21 aminoacids of a human intracellular heme-bindingprotein (HBP, accession number NM 015987) with an amino-terminalacetylation (MW: 2478.28 daltons) (FIGS. 10A and B). For A1, sixpeptides, of which four were fragments of the two longest trypticfragments shown in FIG. 10B, were also consistent with HBP. The porcineHBP was cloned by PCR from liver cDNA using degenerate primers, whichallowed us to confirm the identification, and the perfect conservationof the first 21 aminoacids, as compared to the human sequence. Thissequence was later confirmed following incorporation of porcine HBP ESTsin public databases (accession number AY662687). The two trypticpeptides from A2 covered 50 aminoacids in the amino-terminal domain ofthe 190 aminoacid-long sequence of porcine HBP (FIG. 10C). We assumethat the amino-terminal end of A1 is common to that of A2, although itcould not be demonstrated. The carboxy-terminal end of A1 was notdetermined precisely either, due to the large number of tryptic sitesafter Arg 56 of HBP. The purification was performed three times withdistinct protocols, and the same peptides were identified by massspectrometry in each case.

Comparative Pharmacology and Intracellular Signaling of Formyl PeptideReceptors.

The pharmacology and signaling pathways activated by the three membersof the human FMLP receptor family were investigated in CHO-K1 cellsexpressing the receptors, with or without G alpha 16 and apoaequorin(FIG. 11). The acetylated 21 amino acid peptide, named in Example 8 asF2L (for FPRL2 Ligand), was synthesized and tested in the aequorin-basedassay on these three cell lines, as well as on wild-type CHO-K1 cells,and on CHO-K1 cells expressing ChemerinR and other GPCRs. The syntheticF2L peptide was shown to activate the FPRL2-expressing cells with apotency similar to that of the native peptide purified from spleen, and,with a much lower efficiency, FPRL1 and FPR (see below), but wascompletely inactive on all other cell lines tested (data not shown). F2Lwas also tested in a cAMP accumulation assay on CHO-K1 cells expressingFPRL2 but not G alpha 16. The synthetic peptide was found to inhibit thecAMP accumulation promoted by forskolin, and was unable to stimulatecAMP production by itself. In the same cells, F2L also promotedintracellular calcium release at low nanomolar concentrations (notshown) and induced at picomolar concentrations the phosphorylation ofthe ERK1/2 MAP-kinases (FIG. 11 I). Kinetics study of MAPK activationshowed a maximal phosphorylation at 15 min (FIG. 11J). Calcium signalingwas totally inhibited by Pertussis toxin pretreatment, demonstrating thecoupling of the FPRL2 receptor to the Gi family of heterotrimeric Gproteins (FIG. 11G).

The comparative pharmacology of the three formyl peptide receptors wasthen studied in more detail, using F2L and four reference agonists ofFPR and FPRL1 (FMLP, the hexapeptides WKYMVM (SEQ ID NO: 21) and WKYMVm(SEQ ID NO: 23), and the CCL23-derived SHAAG peptide (Residues 14-18 ofSEQ ID NO: 22)). Concentration-action curves and the resultingfunctional parameters were established both in the aequorin-based assay,and the cAMP accumulation assay following stimulation by 10 μM forskolin(FIGS. 11A and B and Table 1). Among the tested peptides, F2L (SEQ IDNO18) was by far the most potent on FPRL2, with an EC50 of 10 nM in theaequorin assay and 5 nM in the cAMP assay.

F2L appeared also as highly specific, as a weak activity was obtained onFPRL1 (EC50 of 567 and 234 nM in the aequorin and cAMP assays,respectively), while on FPR, only partial inhibition of cAMPaccumulation was obtained for 1 μM F2L, and no activity was detected inthe aequorin assay up to 5 μM. For the other peptides, the EC50 valuesobtained for FPR and FPRL1 (Table 1) were essentially as described inthe literature. However, significant differences with published datawere observed when testing the two W-hexapeptides on theFPRL2-expressing cells. Indeed, micromolar concentrations of thesepeptides were required in order to activate FPRL2 (while active at lownanomolar concentrations on FPR and FPRL1). As described, FMLP and SHAAG(Residues 14-18 of SEQ ID NO: 22) were inactive on FPRL2.

To further confirm that F2L is a specific high affinity ligand forFPRL2, we performed binding experiments. The results show thatsaturation binding assays performed on FPRL 2-expressing CHO-K1 cellsallowed KD of 11.7±4.9 nM to be determined, and a Bmax of roughly 30,000receptors per cell (FIG. 11C) for the modified F2L peptide, bearing acarboxy-terminal tyrosine. Competition binding assays were performedwith F2L, which displayed an 1C50 of 33.4±0.2 nM (FIG. 11D and Table 1).The hexapeptides WKYMVM (SEQ ID NO: 21) and WKYMVm (SEQ ID NO: 23),SHAAG (Residues 14-18 of SEQ ID NO: 22), and FMLP, did not compete forFPRL2 binding up to concentrations of 3 μM (data not shown). We nextconfirmed the specificity of F2L for FPRL2 through binding experimentson FPR and FPRL1-expressing CHO-K1 cells, using [¹²⁵I]WKYMVm (SEQ ID NO:23) as a tracer. The 1C50 values for WKYMVm (SEQ ID NO: 23) were 22.5±7.6 nM on FPRL1 (FIG. 11E), and 98.4±37.4 nM on FPR (FIG. 11F), but nocompetition was observed for F2L up to concentrations of 3 μM.

By analogy with formyl peptides, we investigated the role of theamino-terminal acetylation of F2L. The non-acetylated peptide wassynthesized and shown to display an EC50 for FPRL2 similar to that ofacetylated F2L (21.1±7.6 nM, n=3) (FIG. 11H). We also tested a truncatedF2L variant lacking the first six aminoacids (F2L[7-21]), because mouseintracellular HBP was originally described by Edman sequencing aslacking this N-terminal part (47). This truncated peptide was found tobe totally inactive in aequorin (FIG. 11H) and binding assay (notshown).

Distribution of Human FPRL2.

We investigated the presence of FPRL2 transcripts in various leukocytepopulations by RT-PCR (FIG. 12A). As previously described (41), FPRL2transcripts were the most abundant in monocytes and immature or maturemonocyte-derived DCs. Maturation of DCs was induced by either LPS,LPS+IFN-γ or CD40L for 3 to 24 hours, with no detectable variation ofthe level of expression of FPRL2 transcripts (FIG. 12A and not shown).They were either absent, or present at very low levels in all other cellpopulations tested. Quantitative RT-PCR was performed on a number oftissues, using DCs as reference.

Transcripts were found at low levels in most tissues, and at higherlevels in lymph nodes, small intestine, lung and adipose tissue (FIG.12B).

Monoclonal antibodies also were generated against human FPRL2 by geneticimmunization, and characterized by FACS on CHO-K1 cell lines expressingFPR, FPRL1 or FPRL2 (FIG. 12C). The results indicate that one of thethree monoclonals (1C4) was essentially specific for FPRL2, exhibitingpoor recognition of FPRL1. The two other antibodies (1D2 and 1E1)recognized equally well both receptors. None however cross-reactedsignificantly with FPR. We investigated the ability of the antibodies toblock F2L signaling on FPRL2-expressing CHO-K1 cells. Their blockingproperties appeared however weak, as only partial inhibition of thesignal was obtained with high concentrations (50 μg/ml) of 1C4 antibody(data not shown). These antibodies were used to confirm the presence ofthe receptor at the surface of DCs. The three monoclonals allowed todetect FPRL2 on immature and mature monocyte-derived DCs, although atvariable levels. FPRL2 expression could be detected in 16 out of 24donors. The experiments on one representative donor are displayed inFIG. 12D. We then compared intracytoplasmic and surface expression ofFPRL2 on DCs by performing FACS analysis following permeabilization ofthe cells. We found significant intracellular expression of FPRL2 evenfor donors for which surface expression was very weak or undetectable(FIG. 12E). Additionally, down regulation of cell surface FPRL2 wasobserved when DCs were cultured in presence of 1 μM F2L for 48 hours(not shown). Altogether these data suggest that the variation ofexpression among donors can be attributed to trafficking parameters,such as internalization of the receptor following its stimulation by aligand present in plasma. For 4 tested donors, maturation of DCs by LPSinduced a slight decrease in surface expression of FPRL2 (FIG. 12F).Finally, we evaluated the only variant of FPRL2 described to date(accession number AAA58482), characterized by an aspartic acid tohistidine substitution at position 338. No difference in expression orfunctional response (cAMP inhibition) was detected following transientexpression in HEK cells, as compared to the FPRL2 form used initially(data not shown).

Biological Activity of F2L in Primary Immune Cells.

The biological function of F2L was investigated on leukocytepopulations. By analogy to the role of FPR and FPRL1 in chemoattraction,and given the distribution of FPRL2, we focused on the measurement ofcalcium mobilisation and chemotaxis on monocytes and monocyte-derivedDCs.

The results show that F2L promoted intracellular Ca²⁺ flux in immatureDCs (FIG. 13A), as well as mature DCs (not shown), in a dose-dependentmanner. The amplitude of the response, although variable according toindividuals, was comparable to that resulting from the stimulation by 10nM FMLP (FIG. 13B). Out of 12 donors tested, a strong response wasobtained in 7 cases, a weak response in 2 cases, and no response for 3donors. This is attributed to the variable expression level of FPRL2, asdetermined by FACS analysis. Calcium mobilization was also observed inpurified monocytes, in response to 100 nM F2L, although the amplitude ofthe signal was lower than with DCs (FIG. 13C). Human F2L also promotedex vivo migration of immature DCs and monocytes (FIGS. 13D and E). Cellmigration in response to F2L was mainly due to chemotaxis rather thanchemokinesis as assessed in checkerboard experiments (data not shown).Maximal chemotactic responses were obtained for concentrations of 300 pMto 1 nM. The bell-shaped chemotactic response, with a maximumcorresponding to concentrations below the EC50 derived from otherfunctional assays, is typically observed for other chemotactic factorssuch as chemokines.

In this example, a natural ligand for the receptor FPRL2 has beenidentified. Starting from spleen, F2L has been isolated andcharacterized, as the first natural agonist displaying both highaffinity and high selectivity for FPRL2. F2L binds and activates FPRL2in the low nanomolar range, while the previously described ligands ofthe receptor (annexin I-derived Ac-1-25, bacterial Hp 2-20, andsynthetic W peptides) are essentially FPRL1 agonists displaying weakactivities on FPRL2. It should be noted that the synthetic hexapeptidesWKYMVM (SEQ ID NO: 21) and WKYMVm (SEQ ID NO: 23) were initiallydescribed as high affinity agonists of FPRL2, on the basis ofexperiments conducted on purified leukocyte populations orFPRL2-expressing HL-60 cells (40, 41). Other data contradict theseobservations, describing activities of these peptides in the micromolarrange on FPRL2 expressed in RINm5F (40) or HEK 293 cells (39). These twopeptides effectively required micromolar concentrations to elicitcalcium influx in FPRL2-expressing CHO- K1 cells, while they were activeat low nanomolar concentrations on FPR and FPRL1 expressed in the samesystem.

TABLE 1 Binding and activation of CHO-K1 cells expressing FPRL2, FPRL1or FPR by F2L, FMLP, WKYMVm (SEQ ID NO; 23), WKYMVM (SEQ ID NO: 21) andSHAAG (Residues 14-18 of SEQ ID NO: 22) were studied using a bindingassay, an aequorin-based assay and an assay measuring the inhibition ofcAMP accumulation. pEC₅₀ (sequorin pIC₅₀ (binding Receptor Ligand assay)pEC₅₀ (cAMP assay) assay) FPRL2 F2L 8.02 ± 0.13 (n = 9) 8.24 ± 0.06 (n =4) 7.48 ± 0.003 (n = 3)  WKYMVm <6 NT <6 WKYMVM <6 <6 NT SHAAG <6 <6 <6FMLP <6 <6 <6 FPRL1 F2L 6.26 ± 0.12 (n = 3) 6.65 ± 0.16 (n = 8) <6WKYMVm 10.57 ± 0.10 (n = 3)  NT 7.66 ± 0.15 (n = 3) WKYMVM 10.04 ± 0.18(n = 3)  10.27 ± 0.27 (n = 4)  NT SHAAG 9.27 ± 0.06 (n = 3) 9.23 ± 0.17(n = 6) NT FMLP 5.94 ± 0.03 (n = 3) <6 NT FPR F2L <6 <6 <6 WKYMVm 9.18 ±0.16 (n = 3) NT 7.03 ± 0.16 (n = 3) WKYMVM 7.48 ± 0.08 (n = 3) 8.23 ±0.13 (n = 4) NT SHAAG <6 <6 NT FMLP 9.39 ± 0.33 (n = 3) 10.15 ± 0.08 (n= 4)  NT The EC50 and IC50 parameters of the dose-response curves weredetermined by non-linear regression using the Graphpad Prism software.The results represent the mean pEC50 or pIC50 (−Log values of EC50 orIC50 expressed in Mol/L) ± s.e.m. for at least three independentexperiments (n). NT: not tested.

Example 9 Anti-FPRL2 Monoclonal Antibodies Modulate the IntracellularResponse of Human FPRL2

Aequorine Assays

Functional responses were analyzed by recording the luminescence ofaequorine in human FPRL2 and FPRL1 expressing cells following theaddition of agonists or purified monoclonal antibodies.

In brief, cells were collected from plates with PBS containing 5 mMEDTA, pelleted, resuspend at 5×10⁶ cells/ml in DMEM/F-12 mediumcontaining 0.1% bovine serum albumin, incubated with 5 μM coelenterazineH (molecular probes, Inc. Eugene, Oreg.) for 4 h at room temperature anddiluted in DMEM/F-12 medium at a concentration of 5×10⁵ cells/ml. Cellswhere then mixed in an 96 wells plate with the ligands or purifiedmonoclonal antibodies. The light emission was recorded over 60 sec usinga microlumat Luminometer (EG&G Berthold, microplate luminometer LB 96V).

Experimental Design

Reagents, Ligands and Monoclonal Antibodies (Modulators) Used in Assays.

-   -   Aequorine medium (base line of expressing cells).    -   ATP 20 μM and triton 0.1% (maximum light emission in cells).    -   F2L peptide (SEQ ID NO 18), specific ligand of human FPRL2        receptor as described in (Migeotte et al. (2005) J. Exp. Med,        201: 83-93). Two different batches F2L (1) and F2L (2) have been        used.    -   Humanin (HN(N)) peptide (Phoenix Pharmaceuticals, Inc.) as        described in (Masataka et al. (2004) Biochemical and Biophysical        Research Communications, 324:255-261). This peptide is disclosed        as a ligand for FPRL2 and FPRL1.    -   Purified monoclonal antibodies (Mab):        -   1. FPRL2 145C 4F2 1C4        -   2. FPRL2 422F 2B9 1C11        -   3. FPRL2 422F 2G3 1A10        -   4. unrelated antibody which does not bind to FPRL2 (Control            Mab)

Antibodies were purified with protein A sepharose 4B beads according toAmersham protocol (Armersham Pharmacia Biotech, Uppsala, Sweden).Antibodies FPRL2 145C 4F2 1C4, FPRL2 422F 2B9 1C11 and FPRL2 422F 2G31A10 have been deposited with the BCCM/LMBP Plasmid collection,Department of Molecular Biology, Gent University, Technologiepark 927,B-9052, Gent-Zwijnaarde, Belgium, under the Budapest treaty. They havethe provisional accession numbers LMBP 6404CB (FPRL2 145C 4F2 1C4), LMBP6405CB (FPRL2 422F 2B9 1C11), and LMBP 6406CB (FPRL2 422F 2G3 1A10). Thedates of deposition are Apr. 21, 2005 (LMBP 6404CB/FPRL2 145C 4F2 1C4),and Apr. 28, 2005 (LMBP 6405CB/FPRL2 422F 2B9 1C11, and LMBP6406CB/FPRL2 422F 2G3 1A10).

Reagents, peptidic ligands and monoclonal antibodies have been used atdifferent concentrations in assays as indicated in Tables 2 and 3. Eachdata point is in duplicate. F2L and Humanin peptides were used atconcentrations from 2.5 μM to 0.4 nM and Mab at concentrations from 500ug/ml to 5 ug/ml. Human FPRL1 expressing cells were used as negativecontrol to Mab. Functionality of these cells has been control withHumanin.

TABLE 2 96 wells plate scheme in an aequorine assay using human FPRL2expressing cells. 1 2 3 4 5 6 7 8 9 10 11 12 A Medium ATP 20 μM Triton0.1% B F2L (1) F2L (1) F2L (1) F2L (1) F2L (1) F2L (1) 2.5 × 10⁻⁶M 8.3 ×10⁻⁷M 2.8 × 10⁻⁷M    9.3 × 10⁻⁸M 3.1 × 10⁻⁸M 1 × 10⁻⁸M C F2L (1) F2L (1)F2L (1) F2L (2) F2L (2) F2L (2) 3.4 × 10⁻⁹M 1.1 × 10⁻⁹M 4 × 10⁻¹⁰M 2.5 ×10⁻⁶M 8.3 × 10⁻⁷M 2.8 × 10⁻⁷M   D F2L (2) F2L (2) F2L (2) F2L (2) F2L(2) F2L (2) 9.3 × 10⁻⁸M 3.1 × 10⁻⁸M 1 × 10⁻⁸M  3.4 × 10⁻⁹M 1.1 × 10⁻⁹M 4 × 10⁻¹⁰M E Humanin Humanin Humanin Humanin Humanin Humanin 2.5 ×10⁻⁶M 8.3 × 10⁻⁷M 2.8 × 10⁻⁷M    9.3 × 10⁻⁸M 3.1 × 10⁻⁸M 1 × 10⁻⁸M FHumanin Humanin Humanin Control Mab Control Mab Control Mab 3.4 × 10⁻⁹M1.1 × 10⁻⁹M 4 × 10⁻¹⁰M 500 ug/ml 100 ug/ml 20 ug/ml G M1 M1 M1 M1 M2 M2M2 M2 M3 M3 M3 M3 H 500 100 20 5 300 100 20 5 500 100 20 5 ug/ml ug/mlug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml M1 = mAbFPRL2 145C 4F2 1C4, M2 = mAb FPRL2 422F 2B9 1C11, M3 = mAb FPRL2 422F2G3 1A10.

TABLE 3 96 wells plate scheme in an aequorine assay using human FPRL1expressing cells. 1 2 3 4 5 6 7 8 9 10 11 12 A Medium ATP Triton 20 nM0.1% B Humanin Humanin Humanin Humanin Humanin Humanin 2.5 × 10⁻⁶M 8.3 ×10⁻⁷M 2.8 × 10⁻⁷M 9.3 × 10⁻⁸M 3.1 × 10⁻⁸M 1 × 10⁻⁸M C Humanin HumaninHumanin Control Mab Control Mab Control Mab 3.4 × 10⁻⁹M 1.1 × 10⁻⁹M   4× 10⁻¹⁰M 500 ug/ml 100 ug/ml 20 ug/ml D M1 M1 M1 M1 M2 M2 M2 M2 M3 M3 M3M3 E 500 100 20 5 300 100 20 5 500 100 20 5 ug/ml ug/ml ug/ml ug/mlug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml M1 = mAb FPRL2 145C 4F21C4, M2 = mAb FPRL2 422F 2B9 1C11, M3 = mAb FPRL2 422F 2G3 1A10.Results

The charts in FIGS. 14 and 15 depict the results expressed in RLUcorresponding to the 96-well plates indicated in Table 2.

Observations

F2L peptides activates human FPRL2 expressing cells (B1-D6) and Humaninactivates both human FPRL1 (data not shown) and FPRL2 expressing cells(E1-F6) demonstrating the functionality of cells. Mab FPRL2 422F 2B91C11 (G5-G8 and H5-H8) is clearly able to activate human FPRL2expressing cells in a dose dependent manner. Mab FPRL2 422F 2G3 1A10(G9-G12 and H9-H12) is also able to active these cells but to a lesserextent. The control Mab (F7-F12) does not activate FPRL2 expressingcells. No activation on human FPRL1 expressing cells has been observedwith the Mab panel tested (data not shown).

The above-mentioned observations indicate that Mab FPRL2 422F 2B9 1C11and Mab 422F 2G3 1A10 are able to induce a FPRL2 functional response.These two monoclonal antibodies bind to and activate (as agonist) theFPRL2 receptor.

REFERENCES

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All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1. An isolated heme binding protein (HBP) polypeptide consistingessentially of the amino acid sequence represented by SEQ ID NO:18 orSEQ ID NO:19, wherein said polypeptide specifically binds FPRL2 havingthe amino acid sequence represented by SEQ ID NO:
 2. 2. The isolated HBPpolypeptide of claim 1, wherein the HBP polypeptide is detectablylabeled.
 3. The isolated HBP polypeptide of claim 2, wherein thedetectable label is a moiety selected from the group consisting of aradioisotope, a fluorophore, a quencher of fluorescence, an enzyme, andan affinity tag.
 4. A composition comprising the polypeptide of claim 1.